CN101255406A - Human ovarian cancer drug-resistant cell strain and culture method thereof - Google Patents
Human ovarian cancer drug-resistant cell strain and culture method thereof Download PDFInfo
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- CN101255406A CN101255406A CNA2006101525159A CN200610152515A CN101255406A CN 101255406 A CN101255406 A CN 101255406A CN A2006101525159 A CNA2006101525159 A CN A2006101525159A CN 200610152515 A CN200610152515 A CN 200610152515A CN 101255406 A CN101255406 A CN 101255406A
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Abstract
The invention relates to a TOP medicament resistant cell line human ovarian cancer, with a accession number of CGMCC No 1217, which is selecting human ovarian cancer cell line as parental cell, putting the cell into RPMI1640 culture medium containing 15% calf serum, putting into incubator with 5% CO<SUB>2</SUB> and 37 DEG C to culture; acquiring cells in logarithmic growth phase, counting after digesting with 0.25% pancreatin, inoculating 5*10<SUP>5</SUP>/ml into culture bottle according to amount of 5ml in each bottle, when cells adheres to wall after 24 hours, reacting for 2 hours with a TOP final content of 2160ng/ml, discarding culture medium, washing by phosphate buffer for 3 times, replacing fresh culture medium, second impulsing after growing is resumed, and repeatedly operating, until it is impulsed with medicament for 13 times. The cell line is constructed for 10 months, medicament resistance of cell is 10.67 per cell, P<0.01 comparing with sensitive cell, has cross resistance to mitoxantrone, camptothecin, vincristine besides resistance to TOP.
Description
Technical field
The present invention relates to a kind of selection human oophoroma cell line SKOV3 as parental cell, select for use clinical cancer therapy drug topotecan commonly used as inductor, with intermittently medication of heavy dose of impact mode repeatedly, set up drug resistant cell strain of human oophoroma and the cultural method thereof of topotecan multidrug resistant cell line SKOV3/TOP.
Background technology
Ovarian cancer is one of the highest gynecologic malignant tumor of lethality rate, at present unites to unite as linearize treatment scheme, topotecan and the platinum class of ovarian cancer with taxol and platinum class to be widely used in clinical as the two wires chemotherapy regimen.Yet, often run into the drug-fast problem of these medicines.At present relevant drug-fast research adopts the concentration gradient incremental method at external evoked drug-resistant cell strain more, but this method and clinical chemotherapy repeatedly, the characteristics of medication at intermittence differ greatly.
Summary of the invention
The objective of the invention is to utilize human oophoroma cell line SKOV3 as parental cell, select for use clinical cancer therapy drug topotecan commonly used,, set up topotecan multidrug resistant cell line SKOV3/TOP with intermittently medication of heavy dose of impact mode repeatedly as inductor.
Cell strain of the present invention is cultivated with the following method:
(1) medicine: select topotecan (TOP);
(2) pharmaceutical quantities: according to formula [(60 * D)/(5000)] * 2 * 10
3[5], wherein 60 is average one-tenth body weight for humans (kg), D clinical medicine dose (mg.kg
-1.d
-1), 5000 is average adult blood capacity (ml), 2 is divided by haematocrit 50%, 10
3Change mg into ug, with reference to ovarian cancer patients clinical chemotherapy dosage, TOP 1.2mg/m
2, be that 3 times of common dose are standard with the dosage of clinical high dose chemotherapy, the final concentration of TOP is that 2160ng/ml is as heavy dose of impact dose;
(3) parental cell is cultivated: people's ovarian epithelium JEG-3 SKOV3 cell grows in the RPMI1640 substratum that contains 15% calf serum, inserts 5%CO
2, cultivate in 37 ℃ of incubators;
(4) ovarian cancer drug-resistant cell strain is induced: the cell in vegetative period of taking the logarithm, count after 0.25% the trysinization, with 5 * 10
5/ ml cell inoculation is in culturing bottle (with every bottle of 5ml inoculation), and cell attachment after 24 hours adds final concentration and be the TOP effect 2 hours of 2160ng/ml again, discard nutrient solution, wash 3 times with phosphate buffered saline buffer (PBS), change fresh medium, treat that the cellular-restoring growth carries out impact second time again.Effect so repeatedly, TOP dosing altogether impacts 13 times, lasts 10 months and builds up drug resistant cell strain of human oophoroma SKOV3/TOP, and all resistance experiments are all carried out after cytotostatic was grown back 2 months, and cell cryopreservation was recovered after 3 months.
Cell strain SKOV3/TOP is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCCNo.1217.
Chemical sproof mensuration: survey the resistance index with mtt assay, draw cell growth curve, the apoptosis rate of cells were tested by flow cytometry cell and cell cycle distribution; The Ultrastructural change of transmission electron microscope observing cell.
1. index of cell drug-resistant (RI): SKOV3/TOP resistance index is 10.67 (P＜0.01), and mitoxantrone (MX), camptothecine (CPT), vincristine(VCR) (VCR) are had tangible crossing drug resistant.See Table 1.
Table 1 SKOV3/TOP cell resistance spectrum
**P＜0.01.
*P＜0.05
2 cell growth curves and population doubling time
Cell growth curve and Cell cycles showed, the growing multiplication of two kinds of mdr cells obviously slows down (P＜0.01) than parental cell, sees Fig. 1.
3.P-170 glycoprotein Function detection result
P-170 glycoprotein content among the drug-resistant cell strain SKOV3/TOP obviously increases (P＜0.01), conforms to its resistance index.See Table 3,5 and Fig. 2-3.
Table 3.SKOV3 cell detection results
Date: 23-Nov-04
Table 5.SKO V3/TOP cell detection results
Date: 23-Nov-04
Description of drawings
Fig. 1 cell growth curve
Fig. 2 SKOV3 cell P-170 glycoprotein fluidic cell is measured
Fig. 3 SKOV3/TOP cell P-170 glycoprotein fluidic cell is measured
Embodiment
With the drug-induced SKOV3 cell of topotecan (TOP), set up the SKOV3/TOP drug-resistant cell strain.
Medicine: topotecan (TOP) is Guizhou hanfang Pharmaceutical Co., Ltd's product.
(1) dosage: according to formula [(60 * D)/(5000)] * 2 * 10
3, wherein 60 is average one-tenth body weight for humans (kg), D clinical medicine dose (mg.kg
-1.d
-1), 5000 is average adult blood capacity (ml), 2 is divided by haematocrit 50%, 10
3Change mg into ug, with reference to ovarian cancer patients clinical chemotherapy dosage, TOP 1.2mg/m
2, be that 3 times of common dose are standard with the dosage of clinical high dose chemotherapy, adopting the final concentration of TOP is that 2160ng/ml is as heavy dose of impact dose;
(2) parental cell is cultivated: people's ovarian epithelium JEG-3 SKOV3 cell grows in the RPMI1640 substratum that contains 15% calf serum, inserts 5%CO
2, cultivate in 37 ℃ of incubators.
(3) ovarian cancer drug-resistant cell strain is induced: the cell in vegetative period of taking the logarithm, count after 0.25% the trysinization, with 5 * 10
5/ ml cell inoculation is in culturing bottle (with every bottle of 5ml inoculation), cell attachment after 24 hours, add final concentration again and be the TOP effect 2 hours of 2160ng/ml, discard nutrient solution, wash 3 times, change fresh medium with phosphate buffered saline buffer (PBS), treat that cellular-restoring growth carries out impact second time again, effect so repeatedly, TOP dosing altogether impacts 13 times, lasts 10 months and builds up drug resistant cell strain of human oophoroma SKOV3/TOP.All resistance experiments are all carried out after cytotostatic was grown back 2 months, and cell cryopreservation was recovered after 3 months, and its resistance is still stable.
SKOV3/TOP resistance index is 10.67 (P＜0.01), except that to the TOP resistance, mitoxantrone (MX), camptothecine (CPT), vincristine(VCR) (VCR) is had tangible crossing drug resistant.
The unit of this biological material specimens of preservation: China Committee for Culture Collection of Microorganisms common micro-organisms center
Address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City
Preservation date: 2004.09.02
Deposit number: CGMCC No.1217
Classification name: ovarian cancer drug-resistant cell strain.
Claims (2)
1. drug resistant cell strain of human oophoroma, it is characterized in that: cell strain is called SKOV3/TOP, is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCCNo.1217.
2. the cultural method of a kind of drug resistant cell strain of human oophoroma according to claim 1 is characterized in that:
(1) medicine: topotecan;
(2) dosage: adopting the final concentration of topotecan is that 2160ng/ml is as heavy dose of impact dose;
(3) parental cell is cultivated: people's ovarian epithelium JEG-3 cell grows in the RPMI1640 substratum that contains 15% calf serum, inserts 5%CO
2, cultivate in 37 ℃ of incubators;
(4) ovarian cancer drug-resistant cell strain is induced: the cell in vegetative period of taking the logarithm, count after 0.25% the trysinization, with 5 * 10
5/ ml cell inoculation is in culturing bottle, with every bottle of 5ml inoculation, cell attachment after 24 hours adds final concentration and is the topotecan effect 2 hours of 2160ng/ml again, discards nutrient solution, wash 3 times with phosphate buffered saline buffer, change fresh medium, treat that the cellular-restoring growth carries out second time again and impact, act on so repeatedly, topotecan dosing is altogether impacted 13 times, lasts 10 months and builds up drug resistant cell strain of human oophoroma SKOV3/TOP.
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| CN101255406B CN101255406B (en) | 2010-05-26 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101503673B (en) * | 2009-02-02 | 2011-01-12 | 四川大学华西第二医院 | Cervical cancer multidrug resistance cell strain established by etoposide induction |
| CN101503675B (en) * | 2009-02-02 | 2011-01-12 | 四川大学华西第二医院 | Oophoroma multidrug resistance cell strain established by etoposide induction |
| CN103224910A (en) * | 2013-04-25 | 2013-07-31 | 南京凯基生物科技发展有限公司 | Human ovarian cancer multidrug resistant cell line |
| CN109929804A (en) * | 2017-12-15 | 2019-06-25 | 天津医科大学肿瘤医院 | A kind of abortion syndrome and its preparation method and application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK0572557T3 (en) * | 1991-02-21 | 2010-02-01 | Smithkline Beecham Corp | Use of topotecan in the manufacture of a medicament for use in the treatment of ovarian cancer |
| CN1139655C (en) * | 2001-08-23 | 2004-02-25 | 北京大学人民医院 | Establishment of immortal human ovary carcinoma cell strain |
-
2005
- 2005-04-22 CN CN200610152515A patent/CN101255406B/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101503673B (en) * | 2009-02-02 | 2011-01-12 | 四川大学华西第二医院 | Cervical cancer multidrug resistance cell strain established by etoposide induction |
| CN101503675B (en) * | 2009-02-02 | 2011-01-12 | 四川大学华西第二医院 | Oophoroma multidrug resistance cell strain established by etoposide induction |
| CN103224910A (en) * | 2013-04-25 | 2013-07-31 | 南京凯基生物科技发展有限公司 | Human ovarian cancer multidrug resistant cell line |
| CN109929804A (en) * | 2017-12-15 | 2019-06-25 | 天津医科大学肿瘤医院 | A kind of abortion syndrome and its preparation method and application |
| CN109929804B (en) * | 2017-12-15 | 2020-10-16 | 天津医科大学肿瘤医院 | Human ovarian cancer cell line and preparation method and application thereof |
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