CN106916788B - People's taxol resistance stomach cancer cell HGC-27/PTX and preparation method thereof - Google Patents
People's taxol resistance stomach cancer cell HGC-27/PTX and preparation method thereof Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Abstract
The invention discloses people's taxol resistance stomach cancer cell HGC-27/PTX and preparation method thereof.People's taxol resistance stomach cancer cell HGC-27/PTX provided by the present invention is CGMCC No.13596 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center.People's taxol resistance stomach cancer cell HGC-27/PTX character provided by the invention is stablized, and multiple passage can be stablized, and convenient for furtheing investigate to the molecular mechanism for leading to cells resistance, is the ideal model for studying Patients with Gastric Cancer resistance mechanism, has important application value.
Description
Technical field
The present invention relates to fields of biomedicine, and in particular to people taxol resistance stomach cancer cell HGC-27/PTX and its preparation
Method.
Background technique
Gastric cancer is the third-largest reason of tumor lethal in world wide, the China incidence and mortality Jun Ju malignant tumour
Second.In China, it has been progressive stage or advanced stage when being made a definite diagnosis more than 70% patient, has lost surgical radical treatment chance, drug therapy is
The main means of advanced gastric carcinoma.In gastric cancer three categories chemotherapeutics, taxol has the characteristics quilts such as curative effect is high, tolerance is good
It is widely used in curing gastric cancer.But with the extension of administration time, taxol resistance, which seriously hinders patient, to be continued to benefit.Research is purple
China fir alcohol resistance mechanism and to explore reversing drug resistance strategy be the key that solve the problems, such as, therefore be badly in need of good resistance mechanism diagnostic cast
Type.
Summary of the invention
Technical problem to be solved by the invention is to provide the models of the taxol resistance mechanism of research human gastric cancer.
In order to solve the above technical problems, present invention firstly provides people's taxol resistance stomach cancer cell HGC-27/PTX.
People's taxol resistance stomach cancer cell HGC-27/PTX provided by the present invention, in Chinese microorganism strain preservation pipe
The deposit number of reason committee common micro-organisms center is CGMCC No.13596.
People's taxol resistance stomach cancer cell HGC-27/PTX CGMCC No.13596 is preparing the application in product
It belongs to the scope of protection of the present invention；The function of the product can be A1) or A2) or A3):
A1 resistant models) are used as；
A2 the taxol resistance mechanism of human gastric cancer) is studied；
A3 the drug-resistant phenotype of gastric carcinoma cells) is studied.
The preparation method of people's taxol resistance stomach cancer cell also belongs to protection scope of the present invention.People's taxol resistance gastric cancer
The preparation method of cell may include following steps: using human gastric cancer cell line HGC-27 as parental cell, using taxol induced, obtain
Obtain people's taxol resistance stomach cancer cell.
In above-mentioned preparation method, described " using taxol induced " successively may include following steps:
(1) human gastric cancer cell line HGC-27 is cultivated, cell suspending liquid is obtained；
(2) after completing step (1), taxol induced is carried out, until human gastric cancer cell line HGC-27 can be under taxol just
It is frequently grown；Carry out taxol induced when, paclitaxel concentration by subtract improve.
In above-mentioned preparation method, the culture medium of the culture concretely contains 10% (v/v) fetal calf serum and 1% (v/v)
The RPMI-1640 complete medium of penicillin/streptomycin.
In above-mentioned preparation method, the condition of the culture are as follows: 37 DEG C.
In above-mentioned preparation method, " when carrying out taxol induced, paclitaxel concentration is stepped up " is concretely carried out
When taxol induced, paclitaxel concentration successively can for 2.3nM, 4.6nM, 9.2nM, 18.4nM, 36.8nM, 73.6nM and
149.9nM。
People's taxol resistance stomach cancer cell HGC-27/PTX CGMCC No.13596 is identifying drug to be measured to tumour
Application in resistance also belongs to protection scope of the present invention.
In above-mentioned application, the tumour can be gastric cancer.
People's taxol resistance stomach cancer cell HGC-27/PTX CGMCC No.13596 is in antitumor medicine screening
Using also belonging to protection scope of the present invention.
In above-mentioned application, the anti-tumor drug can be anti-gastric cancer medicament.
People's taxol resistance stomach cancer cell HGC-27/PTX CGMCC No.13596 inhibits tumor proliferation in screening
Application in drug also belongs to protection scope of the present invention.
In above-mentioned application, the tumour can be gastric cancer.
Compared with the prior art, beneficial effects of the present invention are as follows:
(1) human gastric cancer cell line HGC-27 is a good external model.It is clinically used for the chemotherapeutics for the treatment of gastric cancer
Often occur drug resistance problems during use, it is the important original for influencing its chemotherapy and benefiting that patient, which generates drug resistance to drug,
Cause.People's taxol resistance stomach cancer cell HGC-27/PTX character provided by the invention is stablized, and multiple passage can be stablized, convenient for leading
The molecular mechanism of cells resistance is caused to be furtherd investigate.
(2) people's taxol resistance stomach cancer cell HGC-27/PTX provided by the invention has the biology of clinically Patients with Gastric Cancer
Character is learned, is the ideal model for studying Patients with Gastric Cancer resistance mechanism, greatly facilitates to its biological characteristics and drug-resistant phenotype
Variation is studied.
(3) disadvantage of anti-tumor drug be also easy to produce drug resistance and chemotherapy made to fail, and, it is generally the case that certain
Kind mdr cell can usually have crossing drug resistant phenomenon to a variety of drugs, i.e., a kind of drug resistance of drug also results in body to other medicines
The tolerance of object.This jacket cell model that the present invention establishes generates drug resistance to taxol, but to other two gastric cancers Common Chemotherapy medicines
Object (cis-platinum and fluorouracil) has no drug resistance, and is ideal single medicine taxol resistance Mechanism Study model.
Therefore, people's taxol resistance stomach cancer cell HGC-27/PTX CGMCC No.13596 provided by the invention has weight
The application value wanted.
Detailed description of the invention
Fig. 1 is the experimental result of 1 step 2 of embodiment.
Fig. 2 is the experimental result of embodiment 3.
Preservation explanation
Scientific description: people's taxol resistance stomach cancer cell
Join the biomaterial (strain) of evidence: HGC-27/PTX
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism abbreviation: CGMCC
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date: on 2 7th, 2017
Collection is registered on the books number: CGMCC No.13596
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.The experimental materials used in the following example is bought unless otherwise specified
In the qualified conventional biomaterials company of tool.Quantitative experiment in following embodiment is respectively provided with three repeated experiments, as a result takes
Average value.
(product name is " 0.25%Trypsin- for RPMI-1640 culture medium, fetal calf serum, 0.25%EDTA trypsase
EDTA (1 ×) ") and 100 × penicillin/streptomycin be the product of GIBCO company.Phosphate buffer (i.e. PBS buffer solution)
For the product of Hyclone company.Human gastric cancer cell line HGC-27 is Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's cell centre
Product.Paclitaxel injection is the product of Beijing XieHe medicine Factory；Specification is 30mg/5mL.Cisplatin injections are Hospira
The product of Australia Pty Ltd；Specification is 50mg/50mL.Fluorouracil Injection is the product in the pharmaceutical factory Tianjin Jin Yao；Rule
Lattice are 0.25g/10mL.
RPMI-1640 complete medium containing 10% (v/v) fetal calf serum and 1% (v/v) penicillin/streptomycin is by 10 bodies
Product part fetal calf serum, 1 100 × penicillin/streptomycin of parts by volume and 89 parts by volume RPMI-1640 complete mediums mix.
CCK-8 working solution the preparation method comprises the following steps: by 10 parts by volume CCK-8 and 90 parts by volume RPMI-1640 complete mediums
It mixes.CCK-8 is the product of Japanese colleague's chemistry institute.
Relative activity calculation formula:
Resistance index calculation formula:
Resistance index=HGC-27/PTX 503nhibiting concentration/HGC-27/DZ half-inhibitory concentration.
The foundation of embodiment 1, people's taxol resistance stomach cancer cell
One, the foundation of people's taxol resistance stomach cancer cell
People's taxol resistance stomach cancer cell is constructed using the external evoked method of paclitaxel concentration, intermittent action is gradually incremented by.Tool
Body step is successively as follows:
1, HGC-27 suspension is prepared
With 0.25%EDTA trypsin digestion human gastric cancer cell line HGC-27, then it is added and " contains 10% (v/v) tire ox blood
The RPMI-1640 complete medium of cleer and peaceful 1% (v/v) penicillin/streptomycin ", is placed in 37 DEG C, 5%CO2It is cultivated in incubator,
Obtaining cell density is 72 × 105The HGC-27 suspension of a/mL.
2, cultivating system is prepared
Tissue Culture Dish is taken, RPMI-1640 complete medium, HGC-27 suspension and paclitaxel injection is added, obtains
Cultivating system.In the cultivating system, the concentration of human gastric cancer cell line HGC-27 is 36 × 105The concentration of a/mL, taxol is
2.3nM。
3, it cultivates
The cultivating system that step 2 is obtained is placed in 37 DEG C, 5%CO248h is cultivated in incubator, then abandons culture medium, is collected
Cell.
4, it cleans
The cell for taking step 3 to collect is added appropriate PBS buffer solution and cleans 3 times (purpose is to wash away dead cell and remove as far as possible
Influence of the left drug to cell).
5, stablize growth, passage and recovery
The cell of step 4 is taken into, RPMI-1640 complete medium is added, is placed in 37 DEG C, 5%CO2It is trained in incubator
It supports, until cell restore normal growth and (uses 0.25%EDTA trypsin digestion and cell during culture and stablize passage 3 times).
According to above-mentioned steps, by " concentration of taxol is 2.3nM " successively replace with " concentration of taxol is 4.6nM ",
" concentration of taxol is 9.2nM ", " concentration of taxol is 18.4nM ", " concentration of taxol is 36.8nM " " taxol
Concentration is 73.6nM " and " concentration of taxol is 149.9nM ", and other steps are all the same, and it is thin to obtain people's taxol resistance gastric cancer
Born of the same parents HGC-27/PTX.
After testing, when paclitaxel concentration is 149.9nM, people's taxol resistance stomach cancer cell HGC-27/PTX can stablize
Growth, passage and recovery.
People's taxol resistance stomach cancer cell HGC-27/PTX has been preserved in Chinese microorganism strain guarantor on 2 7th, 2017
It hides administration committee's common micro-organisms center (abbreviation CGMCC, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), protects
Hiding number is CGMCC No.13596.The full name of people's taxol resistance stomach cancer cell HGC-27/PTX is people's taxol resistance gastric cancer
Cell HGC-27/PTX CGMCC No.13596, referred to as HGC-27/PTX.
During induction screens and obtains people's taxol resistance stomach cancer cell HGC-27/PTX, by parental cell (i.e. people
Gastric carcinoma cell lines HGC-27) it routine culture and passes on, finally make parental cell and people's taxol resistance stomach cancer cell HGC-27/
PTX has roughly the same passage number.Parental cell after the culture is named as HGC-27/DZ.
Two, morphological observation
The form for observing HGC-27/DZ and HGC-27/PTX respectively under inverted microscope both compares cellular morphology, big
Small and content etc..
Experimental result is shown in Fig. 1 (A be HGC-27/DZ, C and D with B be HGC-27/PTX).The result shows that HGC-27/DZ and
HGC-27/PTX is in monolayer adherence growth；HGC-27/DZ cell size is uniform, sharpness of border, cell are in shuttle shape more；With HGC-
27/DZ is compared, and HGC-27/PTX obviously shortens and is rounded, and has agglomerating aggregation growth tendency, and most cells have short feeler, is had more
Nuclear phenomenon, cytoplasm endoparticle sample substance increase.
Embodiment 2, HGC-27/PTX are to the sensibility of taxol, cis-platinum and fluorouracil
1, sensibility of the HGC-27/PTX to taxol
Experimental group is set, following steps are then carried out:
(1) in the RPMI-1640 complete medium for containing 10% (v/v) fetal calf serum and 1% (v/v) penicillin/streptomycin
Middle culture cell (HGC-27/PTX or HGC-27/DZ) to be measured, obtains cell suspending liquid.
(2) take 96 orifice plates, every hole be inoculated with the preparation of 100 μ L steps (1) cell suspending liquid (every hole containing cell to be measured about 1 ×
104It is a), it is placed in 37 DEG C, 5%CO2It is cultivated for 24 hours in incubator.
(3) after completing step (2), 96 orifice plate is taken, paclitaxel injection is added, obtains system for handling；When to be measured thin
When born of the same parents are HGC-27/PTX, the concentration of the taxol in system for handling is 2.5nM, 5nM, 10nM, 50nM, 250nM or 1250nM
(3 multiple holes are arranged in each paclitaxel concentration)；When cell to be measured is HGC-27/DZ, the concentration of the taxol in system for handling
For 1nM, 2nM, 4nM, 8nM, 16nM, 32nM, 64nM or 128nM (3 multiple holes are arranged in each paclitaxel concentration).
(4) after completing step (3), 96 orifice plate is placed in 5%CO2, cultivate 48h in 37 DEG C of incubators, then discard
Culture medium in each hole, every hole are rapidly added 100 μ L CCK-8 working solutions, continue 5%CO2, 37 DEG C of culture 30min, 1h or 2h.Most
Afterwards using the light absorption value at microplate reader detection 450nm, (incubation time different grouping is pressed by group of the light absorption value between 1.0~2.0
) data calculate HGC-27/PTX degree of cell proliferation.
Blank control group is set: with isometric " containing 10% (v/v) fetal calf serum and 1% (v/v) penicillin/streptomycin
RPMI-1640 complete medium " replaces cell suspending liquid, other same experimental groups of operation.Each paclitaxel concentration is arranged 3 again
Hole.
Cell controls group is set: with isometric " containing 10% (v/v) fetal calf serum and 1% (v/v) penicillin/streptomycin
RPMI-1640 complete medium replaces paclitaxel injection, other same experimental groups of operation.3 multiple holes are arranged in cell suspending liquid.
Using concentration of the taxol in hole as abscissa, relative activity is ordinate, is compared to be measured thin after taxol treatment
The degree of born of the same parents' proliferation.
Experimental result is shown in Table the second row in 1.The result shows that the half-inhibitory concentration (IC50) of HGC-27/PTX is
The half-inhibitory concentration (IC50) of 658.3nM, HGC-27/DZ are 4.73nM；Resistance index (RI) is 139.18.Therefore, HGC-
27/PTX belongs to the resistance to taxol of height, and (be documented: Resistance index < 5 are low drug resistance；5 < Resistance index < 15 are
Etc. drug resistances；Resistance index > 15 are height drug resistance)).
Table 1
Note: * indicates significant difference.
2, sensibility of the HGC-27/PTX to cis-platinum
Experimental group is set, following steps are then carried out:
(1) with (1) in step 1.
(2) with (2) in step 1.
(3) after completing step (2), 96 orifice plate is taken, cisplatin injections are added, obtain system for handling；In system for handling
The concentration of cis-platinum be 0.5nM, 1nM, 2nM, 4nM, 8nM or 16nM (each cis-platin concentrations be arranged 3 multiple holes).
(4) with (4) in step 1.
Blank control group is set: with isometric " containing 10% (v/v) fetal calf serum and 1% (v/v) penicillin/streptomycin
RPMI-1640 complete medium " replaces cell suspending liquid, other same experimental groups of operation.3 multiple holes are arranged in each cis-platin concentrations.
Cell controls group is set: with isometric " containing 10% (v/v) fetal calf serum and 1% (v/v) penicillin/streptomycin
RPMI-1640 complete medium " replaces cisplatin injections, other same experimental groups of operation.3 multiple holes are arranged in cell suspending liquid.
Using concentration of the cis-platinum in hole as abscissa, relative activity is ordinate, compares cell increasing to be measured after cisplatin treated
The degree grown.
Experimental result is shown in Table the third line in 1.The result shows that HGC-27/DZ and HGC-27/PTX to the sensibility of cis-platinum without
Significant difference.
3, sensibility of the HGC-27/PTX to fluorouracil
Experimental group is set, following steps are then carried out:
(1) with (1) in step 1.
(2) with (2) in step 1.
(3) after completing step (2), 96 orifice plate is taken, Fluorouracil Injection is added, obtains system for handling；Handle body
The concentration of fluorouracil in system is 1nM, 2nM, 4nM, 8nM, 16nM, 32nM, 64nM or 128nM (each fluorouracil concentration
3 multiple holes are set).
(4) with (4) in step 1.
Blank control group is set: with isometric " containing 10% (v/v) fetal calf serum and 1% (v/v) penicillin/streptomycin
RPMI-1640 complete medium " replaces cell suspending liquid, other same experimental groups of operation.Each fluorouracil concentration is arranged 3 again
Hole.
Cell controls group is set: with isometric " containing 10% (v/v) fetal calf serum and 1% (v/v) penicillin/streptomycin
RPMI-1640 complete medium " replaces Fluorouracil Injection, other same experimental groups of operation.Cell suspending liquid is arranged 3 again
Hole.
Using concentration of the fluorouracil in hole as abscissa, relative activity is ordinate, compare fluorouracil processing after to
Survey the degree of cell Proliferation.
Experimental result is shown in Table fourth line in 1.The result shows that sensitivity of the HGC-27/DZ and HGC-27/PTX to fluorouracil
Property is without significant difference.
The above results show that HGC-27/PTX generates drug resistance to taxol, but to other two gastric cancers Common Chemotherapy drugs
(cis-platinum and fluorouracil) has no drug resistance, and is ideal single medicine taxol resistance Mechanism Study model.
The cell cycle detection of embodiment 3, HGC-27/PTX
1, in the RPMI-1640 complete medium containing 10% (v/v) fetal calf serum and 1% (v/v) penicillin/streptomycin
It cultivates cell to be measured (HGC-27/PTX or HGC-27/DZ), obtains cell suspending liquid first.
2, take 6 orifice plates, the cell suspending liquid first of every hole inoculation 2mL step (1) preparation (every hole containing about cell to be measured 30 ×
104It is a), it is placed in 37 DEG C, 5%CO2It is cultivated for 24 hours in incubator.
3, the 6 orifice plates are taken, paclitaxel injection is added, obtains system for handling；When cell to be measured is HGC-27/PTX,
The concentration of taxol in system for handling is 128nM, 200nM or 400nM (3 multiple holes are arranged in each paclitaxel concentration)；When to
When survey cell is HGC-27/DZ, the concentration of the taxol in system for handling is 1nM, 2nM or 4nM (each paclitaxel concentration setting
3 multiple holes).
4, after completing step 3, the 6 orifice plates are placed in 5%CO2, cultivate 48h in 37 DEG C of incubators, obtain cell and suspend
Liquid second.
5, cell suspending liquid first or cell suspending liquid second are taken, room temperature 1000rpm/min is centrifuged 5min, collects precipitating.
6, take step 5 collect precipitating, be added 1mL pre-cooling PBS buffer solution be resuspended, then room temperature 1000rpm/min from
Heart 5min collects precipitating.
7, the precipitating for taking step 6 to collect, the PBS buffer solution that 250 μ L pre-cooling is added are resuspended, and it is pre- that 750 μ L are then added dropwise
Cold dehydrated alcohol (shaking when being added dropwise), mixes, is placed in 4 DEG C of refrigerator overnights and fixes.
8, after completing step 7, room temperature 1000rpm/min is centrifuged 5min, collects precipitating.
9, the precipitating for taking step 8 to collect is added 1mL PBS buffer solution and is resuspended, and room temperature 1000rpm/min is centrifuged 5min, receives
Collection precipitating.
10,300 μ L PI/RNase buffers are added in the precipitating for taking step 9 to collect, and room temperature, which is protected from light, is incubated for 15min；Cell
The flow cytomery cell cycle is used after the screen to filtrate, then with point of 5 software of Graphpad Prism analysis cell cycle
Cloth.
Experimental result is shown in Fig. 2.The result shows that compared with HGC-27/DZ, the S phase (41.44% of HGC-27/PTX
Vs.32.9%, P < 0.01) it increased significantly with the ratio of G2/M phase cell (37.87%vs.18.36%, P < 0.01)；With purple
When China fir alcohol handles HGC-27/DZ, G2/M phase cell proportion increases with paclitaxel concentration and is increased；With taxol treatment HGC-27/
When PTX, the influence of taxol cell cycle is smaller, illustrates that HGC-27/PTX generates drug resistance to taxol.
Claims (7)
1. people taxol resistance stomach cancer cell HGC-27/PTX, in China Committee for Culture Collection of Microorganisms's commonly micro- life
The deposit number at object center is CGMCC No.13596.
2. people's taxol resistance stomach cancer cell HGC-27/PTX CGMCC No.13596 is in preparing product described in claim 1
Application；The function of the product be A1) A2) or A3):
A1 resistant models) are used as；
A2 the taxol resistance mechanism of human gastric cancer) is studied；
A3 the drug-resistant phenotype of gastric carcinoma cells) is studied.
3. the preparation method of people's taxol resistance stomach cancer cell, includes the following steps: with human gastric cancer cell line HGC-27 for parent
Cell obtains people's taxol resistance stomach cancer cell using taxol induced.
4. preparation method as claimed in claim 3, it is characterised in that: described " using taxol induced " successively includes following step
It is rapid:
(1) human gastric cancer cell line HGC-27 is cultivated, cell suspending liquid is obtained；
(2) after completing step (1), taxol induced is carried out, until human gastric cancer cell line HGC-27 can normally give birth under taxol
It is long；When carrying out taxol induced, paclitaxel concentration is gradually increased.
5. people's taxol resistance stomach cancer cell HGC-27/PTX CGMCC No.13596 described in claim 1 is identifying medicine to be measured
Object is to the application in tumor resistance；The tumour is gastric cancer.
6. people's taxol resistance stomach cancer cell HGC-27/PTX CGMCC No.13596 described in claim 1 is antitumor in screening
Application in drug；The anti-tumor drug is anti-gastric cancer medicament.
7. people's taxol resistance stomach cancer cell HGC-27/PTX CGMCC No.13596 described in claim 1 inhibits swollen in screening
Application in the drug of tumor proliferation；The tumour is gastric cancer.
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