CN103224910A - Human ovarian cancer multidrug resistant cell line - Google Patents
Human ovarian cancer multidrug resistant cell line Download PDFInfo
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Abstract
According to the invention, human ovarian cancer cell line HO-8910 is adopted as an induction object; with a high-dose impact method, and with an in-vitro induction method with gradually increased VP16 concentration and intermittent action, the human ovarian cancer multidrug resistant cell line HO-8910/VP16 is established. The cell ling is preserved at China General Microbiological Culture Collection Center on December 24th, 2012, and has a preservation number of CGMCC NO.7052. The cell line provided by the invention has typical multidrug resistance. With the cell line, an experiment basis is provided for further researching resistance reversal approaches.
Description
Technical field
The invention belongs to the cell engineering field, relate to a kind of human cell's strain, be specifically related to a kind of human ovarian cancer multidrug resistance cell strain and establishment method thereof.
Background technology
In the female sex organ malignant tumour, the ovarian cancer sickness rate occupies the 3rd, and case fatality rate occupies the 1st.This is because the morbidity of most of ovarian cancer is hidden, and lacks method of early diagnosis effectively accurately, and about 70%～80% patient belongs to late period once making a definite diagnosis.
Chemicals is one of important means of treatment human malignancies, yet further improves owing to tumor cell drug resistance has usually limited curative effect, and finally makes the treatment failure.Tumor multi-medicine drug-resistant (multidrug resistance, MDR) be that tumour cell is avoided the important cells defense mechanism that medicine is attacked, be meant that tumour cell also produces the phenomenon of crossing drug resistant to the other drug different with its structure-irrelevant, mechanism of action to the drug-fast while of a kind of chemotherapeutics.Overcome multi-drug resistance of the tumor, seek the new way of reversion of malignant tumor cell multidrug resistance, the raising tumour cell is one of important topic of current tumor area to the susceptibility of chemotherapy.Successfully setting up the drug-resistant tumor cell model is the prerequisite and the basis of carrying out this research.
The establishment method of oncocyte persister has two kinds usually, and the one, progressively increase drug level, successive induction method, the 2nd, heavy dose of ballistic method.Foundation by drug-resistant cell strain is tumor drug resistance model inside and outside the construct further, thus further investigation MDR resistance mechanism, even find the resistance mechanism that other are new.The inside and outside tumor drug resistance model of the foundation by drug-resistant cell strain can be used for the screening of antitumor drug.
Find by retrieval, at present both at home and abroad also not about with human oophoroma cell line HO-8910 for inducing object, set up the bibliographical information of human ovarian cancer multidrug resistance cell strain HO-8910/VP16.
Summary of the invention
In view of the deficiencies in the prior art, the object of the present invention is to provide a kind of human ovarian cancer multidrug resistance cell strain and establishment method thereof.Cell strain of the present invention has typical multidrug resistance characteristic, provides experiment basis for further studying the drug resistance inversion approach.
The object of the present invention is achieved like this:
The present invention adopts human oophoroma cell line HO-8910 for inducing object, induces early stage and adopts heavy dose of ballistic method, later stage to induce employing successive induction method, has set up human ovarian cancer multidrug resistance cell strain HO-8910/VP16.This cell strain is preserved in Chinese typical culture collection center on December 24th, 2012, and preserving number is CGMCC NO.7052.
Particularly, the human ovarian cancer multidrug resistance cell strain HO-8910/VP16 that the present invention relates to sets up acquisition as follows:
(1) human oophoroma cell line HO-8910 is with nutrient solution (the RPMI-1640 nutrient solution that contains 10% calf serum), 5%CO2, under 37 ℃ of conditions, when being cultured to 70%～90% adherent rate, remove supernatant, add VP161000ng/ml and impact cultivation 1 hour, remove the VP16100ng/ml nutrient solution, after the blank RPMI-1640 nutrient solution washing 2 times, change multiplication culture liquid and cultivate 72-96h amplification survivaling cell.When survivaling cell is expanded to 70%～90% adherent rate, repeat again to impact cultivation 1 hour 9 times with the VP161000ng/ml nutrient solution, promptly finish 10 Fluracil 1000ng/ml taxol nutrient solutions altogether and impact cultivation 1 hour, further based on this cell, 1000ng/ml continues to cultivate 7 days.
(2) repeat aforesaid operations, change screening conditions into VP16200ng/ml, 400ng/ml, 800ng/ml nutrient solution successively.
The present invention is by Rh-123 research and the mensuration of cell cycle in morphological observation, growth curve and population doubling time mensuration, drug sensitivity test, the cell, the biological characteristics of appraiser's oophoroma multidrug resistance cell strain HO-8910/VP16, the result has successfully set up the HO-8910/VP16 persister, the resistance index is 81.602, and cis-platinum (cDPP), taxol (Taxol), vincristine(VCR) (YCR), Fluracil (5-FU) and Zorubicin (Adr) have been produced tangible cross resistance.
Description of drawings
Fig. 1 is the cell growth curve figure of HO-8910, HO-8910/VP16.
Fig. 2 is the cell cycle figure of HO-8910 cell strain
Fig. 3 is the cell cycle figure of HO-8910/VP16 cell strain
Fig. 4 is that rhodamine 123 is at the intracellular spirogram that contains of HO-8910
Fig. 5 is that rhodamine 123 is at the intracellular spirogram that contains of HO-8910/VP16
Fig. 6 is the microgram of HO-8910/VP16 cell strain of the present invention.
Embodiment
Further specify the present invention below by embodiment.Embodiments of the invention are only used for illustrating the present invention, rather than limitation of the present invention, and the simple modifications to the inventive method under design prerequisite of the present invention all belongs to the scope of protection of present invention.
Embodiment 1 human ovarian cancer multidrug resistance cell strain HO-8910/VP16 induces foundation
Adopt the external evoked method that progressively increases progressively VP16 concentration, intermittent action to set up human ovarian cancer multidrug resistance cell strain HO-8910/VP16, concrete steps are as follows:
(1) human oophoroma cell line HO-8910 is with nutrient solution (the RPMI-1640 nutrient solution that contains 10% calf serum), 5%CO2, under 37 ℃ of conditions, when being cultured to 70%～90% adherent rate, remove supernatant, add VP161000ng/ml and impact cultivation 1 hour, remove the VP16100ng/ml nutrient solution, after the blank RPMI-1640 nutrient solution washing 2 times, change multiplication culture liquid and cultivate 72-96h amplification survivaling cell.When survivaling cell is expanded to 70%～90% adherent rate, repeat again to impact cultivation 1 hour 9 times with the VP16100ng/ml nutrient solution, promptly finish 10 Fluracil 1000ng/ml taxol nutrient solutions altogether and impact cultivation 1 hour, further based on this cell, 100ng/ml continues to cultivate 7 days.
(2) repeat aforesaid operations, change screening conditions into VP16200ng/ml, 400ng/ml, 800ng/ml nutrient solution successively.
(3) sensitive cells is dead gradually, and the mdr cell continued growth.Induce, change liquid so repeatedly, go down to posterity, last June approximately, finally obtain the persister of stable growth propagation in 800ng/mL VP16, called after HO-8910/VP16.This cell cryopreservation was recovered after February, and the original resistance of still basic maintenance repeatedly more than February goes down to posterity in the nutrient solution that does not contain 5-FU.
Embodiment 2 persister morphological observations
The human ovarian cancer multidrug resistance cell strain HO-8910/VP16 that takes the logarithm vegetative period, observation of cell form under inverted microscope, result as shown in Figure 6: cellular form becomes big, the nuclear shrinkage, cell is easily piled up agglomerating.
Embodiment 3 cell growth curves are measured
Cell dissociation, count, be mixed with the cell suspension that concentration is 5 * 103/mL, every hole adds 100 μ L cell suspensions (500 cells in every hole) in the 96 porocyte culture plates; 6 porocyte culture plates place 37 ℃, cultivate 2d, 4d, 6d, 8d, 10d in the 5%CO2 incubator respectively; 96 orifice plates are carried out MTT dyeing, and λ=490nm measures the OD value; Every hole adds 20 μ L MTT(5mg/mL), continue to cultivate 4 hours at incubator; Discard substratum, every hole adds 150 μ L DMSO dissolving, and shaking table 10 minutes is mixing lightly; λ=490nm, the OD value that microplate reader is read every hole is that X-coordinate, OD value are ordinate zou with time, draws cell growth curve.The cell growth curve of HO-8910, HO-8910/VP16 is seen Fig. 1.
Embodiment 4, PI staining detect the cell cycle
With 0.25% trysinization logarithmic phase cell, the adjusting cell density is 2 * 105/ml, is inoculated in 6 well culture plates, cultivates 24h; Cell is collected in 0.25% trysinization; With the 0.01M PBS washed cell of 4 ℃ of preservations 3 times, 70% ethanol is fixing 5min on ice, and the centrifugal 5min of 2000rpm room temperature is resuspended in cold PBSlml, and is standby; Detect with flow cytometer, excitation wavelength is 488nm, and the reception wavelength is 575nm.The result shows, HO-8910G1 phase, S phase, G2 phase cell are respectively 58.31%, 23.64%, 18.05%, HO-8910/VP16G1 phase, S phase, G2 phase cell are respectively 55.99%, 36.51%, 14.39%, HO-8910/VP16G1 phase, G2 phase cell reduce, S phase cytosis the results are shown in Figure 2, Fig. 3.
Embodiment 5 flow cytometers detect rhodamine 123 and accumulate
With 0.25% trysinization logarithmic phase cell, the adjusting cell density is 2 * 105/ml, is inoculated in 6 well culture plates, cultivates 24h; Cell is collected in 0.25% trysinization; With the 0.01M PBS washed cell of 4 ℃ of preservations 3 times, collecting and adjusting cell concn is 1 * 10
6/ ml, it is the rhodamine 123500uL of 5ug/ml that every pipe adds concentration, 37 ℃ of insulation 30min, the centrifugal 2min of 500rpm, remove nutrient solution, use the extracellular rhodamine of new nutrient solution flush away again, at 37 ℃ of insulation 10min, P-gp glycoprotein function is brought into play, medicine is pumped, wash 1 time with nutrient solution again, be placed in the ice-cold nutrient solution to be detected, the flow cytometer exciting light of 488nm, the test cell fluorescence intensity.The results are shown in Figure 4, Fig. 5.The HO-8910 cell fluorescence intensity is 7105, and the HO-8910/VP16 cell fluorescence intensity is 105, and the intracellular cell rhodamine 123 of HO-8910/VP16 reduces than HO-8910.
Embodiment 6 drug sensitivity tests
Cell dissociation, to count, be mixed with concentration be 1 * 10
5The cell suspension of individual/ml, every hole adds 100ul cell suspension (every hole 1 * 10 in the 96 porocyte culture plates
4Individual cell); 96 porocyte culture plates place 37 ℃, 5%CO
2Cultivated 24 hours in the incubator; To desired concn, every hole adds the corresponding pastille substratum of 100 μ L with perfect medium dilution medicine, and (4) 96 porocyte culture plates place 37 ℃, 5%CO
2Cultivated 24 hours in the incubator; 96 orifice plates are carried out MTT dyeing, and λ=490nm measures the OD value; Count each group inhibiting rate and medicine IC50.
Test-results is referring to table 1.According to the result of table 1 as can be seen, HO-8910/VP16 is 81.602,5.370,5.746,1.959,16.346,3.153 to the resistance index of Etoposide, Fluracil, cis-platinum, taxol, vincristine(VCR), Zorubicin.
Table 1 cell strain is to the I C50 and the resistance index of various chemotherapeutics
| Chemotherapeutics | IC50(ug/ml)HO-8910 | IC50(ug/ml)HO-8910/VP16 | RI |
| Etoposide (VP16) | 0.665 | 54.265 | 81.602 |
| Cis-platinum (cDPP) | 1.215 | 6.524 | 5.370 |
| Taxol (Taxol) | 0.059 | 0.339 | 5.746 |
| Vincristine(VCR) (YCR) | 0.049 | 0.096 | 1.959 |
| Fluracil (5-FU) | 1.182 | 19.321 | 16.346 |
| Zorubicin (Adr) | 0.124 | 0.391 | 3.153 |
Claims (1)
1. human ovarian cancer multidrug resistance cell strain HO-8910/VP16, preserving number is CGMCC NO.7052.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805057A (en) * | 2015-05-04 | 2015-07-29 | 哈尔滨医科大学 | Ovarian cancer multi-drug resistant cell line established by inducing triptolide, and application of cell line |
| CN105368783A (en) * | 2015-10-30 | 2016-03-02 | 温州医科大学附属第一医院 | Human lung cancer paraquat-resistant cell strain |
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| CN101255406A (en) * | 2005-04-22 | 2008-09-03 | 李红霞 | Human ovarian cancer drug-resistant cell strain and culture method thereof |
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| CN1687405A (en) * | 2005-04-22 | 2005-10-26 | 李红霞 | Drug resistant cell strain of human oophoroma and culturing method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805057A (en) * | 2015-05-04 | 2015-07-29 | 哈尔滨医科大学 | Ovarian cancer multi-drug resistant cell line established by inducing triptolide, and application of cell line |
| CN104805057B (en) * | 2015-05-04 | 2018-03-09 | 哈尔滨医科大学 | Oophoroma multidrug resistance cell strain that triptolide induction is established and application thereof |
| CN105368783A (en) * | 2015-10-30 | 2016-03-02 | 温州医科大学附属第一医院 | Human lung cancer paraquat-resistant cell strain |
| CN105368783B (en) * | 2015-10-30 | 2019-05-10 | 温州医科大学附属第一医院 | A kind of human lung cancer cell strain of resistance to paraquat |
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Address after: 210012, building 6, building 18, No. four, LAN Lu, Jiangning District, Jiangsu, Nanjing Patentee after: Jiangsu Kaiji biological technology Limited by Share Ltd Address before: 210012, No. 439, Changhong Road, Yuhua District, Jiangsu, Nanjing Patentee before: Kaiji Biotech Development Co., Ltd., Nanjing |