CN1563412A - Method for determining drug susceptibility to epithelial cancer of ovary through activity telomere enzyme - Google Patents
Method for determining drug susceptibility to epithelial cancer of ovary through activity telomere enzyme Download PDFInfo
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- CN1563412A CN1563412A CN 200410018808 CN200410018808A CN1563412A CN 1563412 A CN1563412 A CN 1563412A CN 200410018808 CN200410018808 CN 200410018808 CN 200410018808 A CN200410018808 A CN 200410018808A CN 1563412 A CN1563412 A CN 1563412A
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- telomerase activation
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Abstract
This invention relates to a method for judging drug sensitivity to ovarian epithelial cancer with telomere zyme activity, first of all, primary culture is carried out to the ovarian epithelial cancer organization, then its solution is extracted to test its primary telomere zyme activity, then to extract the cells after primary culture to be added with cisplatin or adriamycin and continuring the culture, the caner cell telomere zyme activity after processed with drugs is tested to see the decline degree to the primary one by computation and see if the drug is sensitive.
Description
[technical field]
The invention belongs to the diagnosis of malignant tumor field, particularly a kind of method of judging the epithelial ovarian cancer drug susceptibility with telomerase activation.
[background technology]
Cell subtracts the art of going out, and to add postoperative be a kind of conventional treatment pattern of epithelial ovarian cancer based on the combined chemotherapy of cis-platinum.The selection of clinical chemotherapeutics still has certain blindness at present, because the epithelial ovarian cancer chemotherapy regimen comes out according to clinical empirical summary mostly, be primarily aimed at different pathological types and select different chemotherapy regimens, rather than select medication with characteristic according to the biology essence of patient tumors.Therefore will produce different curative effects with a kind of chemotherapy regimen.In order to overcome this problem, prior art is carried out external susceptibility detection to tumour and has been done big quantity research, and method wherein has tetramethyl-azo azoles salt (MTT) detection method, viable count method etc., and the responsive chemotherapeutics of selection of clinical is had certain directive function.But in primary cell culture, because the cellular constituent complexity often be mingled with many normal cells in tumour cell, and above method can not be distinguished the difference of chemotherapy drug effect between tumour cell and normal cell.Therefore, has bigger limitation with it as the drug screening index.In recent years, discover in addition that epithelial ovarian cancer Telomerase Activity more than 90% strengthens, and in normal ovarian tissue, Telomerase often be negative or activity lower, but have not yet to see with the report of telomerase activation as detection of drugs susceptibility index.
[summary of the invention]
The purpose of this invention is to provide a kind of method of judging the epithelial ovarian cancer drug susceptibility with telomerase activation, it is a kind of to detect medication front and back end granzyme activity change as the method for judging the epithelial ovarian cancer chemotherapy drug susceptibility, compare with mtt assay and to have the wider clinical application prospect, can instruct the selection of clinical medication objectively.
The scheme that the present invention is adopted for achieving the above object is a kind of method with telomerase activation judgement epithelial ovarian cancer drug susceptibility, it is characterized in that finishing according to the following steps:
1) get patient's ovarian epithelial cancerous tissue carry out former be commissioned to train foster;
2) get former being commissioned to train and support the original telomerase activation of back tumour cell extracting solution mensuration tumour cell;
3) get the former foster back tumour cell of being commissioned to train and add cis-platinum or Zorubicin continuation cultivation, with the adherent tumour cell of tryptic digestion, tumour cell telomerase activation after the mensuration medication;
4) calculate the decline degree of the original telomerase activation of Telomerase specific activity after the medication, be judged as medicaments insensitive when telomerase activation descends when surpassing 30%, it is insensitive to be lower than at 30% o'clock and to be judged as medicine.
The invention has the beneficial effects as follows: the present invention strengthens according to the epithelial ovarian cancer Telomerase Activity, and in normal ovarian tissue, Telomerase often is negative or active lower character.Handle the epithelial ovarian cancer primary cultured cell with different chemotherapeutics, measure the change of tumour cell extracting solution Telomerase Activity before and after handling, can objectively respond out the sensitivity of tumour cell to chemotherapeutics, active change does not exert an influence and Normocellular existence is to Telomerase.So experimental result is reliable, high specificity, thus instruct the selection of clinical medication effectively.
Experimental results show that: the corresponding medicaments insensitive tumour cell Telomerase Activity of drug-resistant tumor cell Telomerase Activity increases 27.68%～69.06%.Ovarian cancer SKOV3 cell quantity and telomerase activation are proportionate, and promptly ovarian cancer viable cell quantity is many more, and telomerase activation is high more.In the drug susceptibility tumour cell, telomerase activation decline all is higher than 50% before and after the medication, and in corresponding drug-resistant tumor cell, telomerase activation decline all is lower than 30% before and after the medication, illustrates that telomerase activation decline degree is relevant with tumour medicine susceptibility.
The present invention examines side by telomerase activation, and mtt assay is measured inhibition rate of tumor growth, and compares proof with clinical efficacy: the telomerase activation descent method is than the clinical coincidence rate height of mtt assay.
In 11 routine epithelial ovarian cancers, add cis-platinum, Zorubicin respectively and carry out telomerase activation inspection side before and after the medication, the result shows that telomerase activation decline has 9 examples (81.82%), 9 examples (81.82%) respectively 30% above person, and being reduced to 30% under the telomerase activation has 2 examples (18.18%), 2 examples (18.18%) respectively.
Measure inhibition rate of tumor growth with mtt assay, in 11 routine patients, cis-platinum, Zorubicin growth inhibition ratio have 4 examples (36.36%), 7 examples (63.64%) respectively 30% above person, and growth inhibition ratio is lower than 30% 7 examples (63.64%), 4 examples (36.36%) are arranged respectively.
Compare with clinical efficacy, measure epithelial ovarian cancer with telomerase activation descent method and mtt assay cis-platinum and Zorubicin susceptibility and clinical efficacy coincidence rate are respectively 90.91%, 63.64% and 90.91%, 72.73%.
[embodiment]
Judge the method for epithelial ovarian cancer drug susceptibility with telomerase activation, at first will to patient's ovarian epithelial cancerous tissue carry out former be commissioned to train foster, get former being commissioned to train then and support the original telomerase activation of back tumour cell extracting solution mensuration tumour cell, other gets and continues to cultivate 72 hours after the former foster back tumour cell of being commissioned to train adds cis-platinum or Zorubicin, tumour cell telomerase activation after the mensuration medication, use formula again: telomerase activation before telomerase activation/medication after the Δ A=1-medication, the decline degree of the original telomerase activation of Telomerase specific activity after the calculating medication.Be judged as medicaments insensitive when telomerase activation descend to surpass 30%, it is insensitive to be lower than at 30% o'clock and to be judged as medicine.
The epithelial ovarian cancer tissue in primary culture method that the present invention adopts is: the cancerous tissue that excises in the epithelial ovarian cancer corrective surgery is put into immediately the sterile culture bottle that contains the RPMI1640 substratum, in super clean bench, open and put into sterile petri dish, with scissors tissue is shredded after washing down blood repeatedly with physiological saline, and wash repeatedly with RPMI1640.Digested 10～20 minutes down at 37 ℃ with 0.125%～0.25% trypsinase then, make single cell suspension to blow and beat repeatedly after the foetal calf serum deactivation, 1500bpm is centrifugal, abandons supernatant liquor, add 10% foetal calf serum with the cell precipitation spin-up with RPMI1640, adjusting cell concn is 1 * 10
6/ ml is inoculated in the culture dish, at 37 ℃, 5%CO
2Cultivate in the incubator.
The Telomerase activity method that this patent adopts: the TRAP-PCR-ELISA method detects telomerase activation, is semi-quantitative method.Wherein:
1) at first measure protein content in the epithelial ovarian cancer tissue extract by Folin-phenol reagent process (Lowry method):
1. formulate typical curve: get human serum albumin 5mg and be dissolved in the 5ml distilled water, make the working fluid that concentration is 250 μ g/ml again.Get working fluid 0ml, 0.1ml, 0.2ml, 0.4ml, 0.6ml, 0.8ml and 1.0ml respectively, water is supplied 1.0ml.Each concentration is respectively got 3 groups.Add 5ml reagent first in each test tube, mixing in 20～25 ℃ of placements 10 minutes, adds 0.5ml reagent second (FolinShi reagent) again, and vibration immediately shakes up, 20～25 ℃ of insulations 30 minutes, then in 500nm place colorimetric.Replace sample as blank with 1ml distilled water.The drawing standard curve calculates regression equation;
2. testing sample epithelial ovarian cancer tissue extract is carried out above-mentioned detection colorimetric in the same way, and do contrast, bring the result into linear regression equation, protein content in the calculation sample in each capable 0.4ml working fluid;
2) there is not the preparation of RNA enzyme (RNase) experiment condition: handle Eppendorf pipe, thin-walled PCR pipe and various suction nozzle with 0.1% burnt ethylene two fat (DEPC), and the distilled water preparation phosphoric acid buffer of handling with 0.1%DEPC (PBS), with 15 pounds of 20 minutes autoclavings, remove residual DEPC simultaneously again.
3) TRAP-PCR-ELISA detects: collect the tumour cell of digestion back centrifugation, be placed in the Eppendorf pipe, add lysate 200 μ l, ice bath 30 minutes.4 ℃, centrifugal 20 minutes of 13000g gets supernatant liquor and moves in another pipe.Measure with aforesaid method and to get 6 μ g albumen behind the protein content in the tissue extract and carry out PCR, reaction system is 50 μ l, comprising Telomerase substrate, telomeric sequence Auele Specific Primer: P1-TS mark vitamin H primer and P2 amplimer etc.
Then, get positive control solution and negative controls 3 μ l respectively in contrast.Add reaction of degeneration liquid 20 μ l and pcr amplification product 5 μ l, room temperature was placed 10 minutes.Add hybridization buffer 225 μ l, get 100 μ l and change microwell plate over to, hybridization is 2 hours on 37 ℃, 300bpm shaking bath.With lavation buffer solution 250 μ l washing 2 times, add Anti-DIG-POD (being combined with the anti-digoxine antibody of peroxidase) 100 μ l, final concentration 10mU/ml placed 30 minutes under the room temperature, with lavation buffer solution 250 μ l washing 2 times.Add tmb substrate 100 μ l, placed 10-20 minute under the room temperature.Adding stop buffer 100 μ l, is to measure absorbancy under 450nm, the 690nm at wavelength, calculates telomerase activation according to following formula.
△ A=sample value (A
450-A
690)-standard negative control value (A
450-A
690)
Standard negative control value (A wherein
450-A
690) should be less than 0.25U, standard positive control value (A
450-A
690) should be greater than 1.5U, when △ A＞0.2U is judged as the positive.
Measuring medication front and back tumour cell telomerase activation in the present invention, is earlier the ovarian epithelial cancerous tissue to be made single cell suspension with the tryptic digestion method, and adjusting tumour cell concentration is 1 * 10
6/ ml gets 2ml and measures original telomerase activation.Get 2ml inoculation culture ware then in addition, continue cultivation 12 hours, add cis-platinum 10 μ g/ml or Zorubicin 1 μ g/ml, continue cultivation and stopped in 72 hours, with the adherent tumour cell of 0.25% tryptic digestion, telomerase activation after the mensuration medication.
In example 1:53 year, the right serous cystadenocarcinoma of ovary III phase, the row cell subtracts the art of going out, and the remaining focus of postoperative is 1cm, cancerous tissue is commissioned to train fosterly so that tryptic digestion method row is former, because tumour is cauliflower form, matter is poor crisp, so trypsinase concentration selects 0.125%, 37 ℃ to digest 10 minutes down, digests more complete.The detection telomerase activation is 2.495U, add with detecting telomerase activation behind cis-platinum, the Zorubicin and be respectively 1.254U, 1.023U, telomerase activation descends 49.74%, 59.0% respectively before and after the medication, point out this patient all responsive to cis-platinum, Zorubicin, and the inhibition rate of tumor cell of mtt assay mensuration cis-platinum, Zorubicin is respectively 10.33%, 48.67%, the prompting tumour is insensitive to cis-platinum, and to the Zorubicin sensitivity, clinical follow up results shows that this patient is effective to cis-platinum, endoxan scheme chemotherapy.
Example 2: patient 58 years old, the both ovaries serous papillary adenocarcinoma III phase, the row cell subtracts the art of going out, the remaining focus of postoperative is 1cm, cancerous tissue with tryptic digestion method row former be commissioned to train foster because tumor tissues is nodositas, organize fine and close, so trypsinase concentration selects 0.25%, 37 ℃ to digest 20 minutes down, digests more complete.The detection telomerase activation is 2.705U, add with detecting telomerase activation behind cis-platinum, the Zorubicin and be respectively 1.632U, 1.569U, telomerase activation descends 39.67%, 42.0% respectively before and after the medication, point out this patient all responsive to cis-platinum, Zorubicin, and the inhibition rate of tumor cell of mtt assay mensuration cis-platinum, Zorubicin is respectively 14.45%, 34.91%, the prompting tumour is insensitive to cis-platinum, and to the Zorubicin sensitivity, clinical follow up results shows that this patient is effective to cis-platinum, endoxan scheme chemotherapy.But patient's recurrence in 1 year behind 8 course of treatment end of chemotherapy, blood CA
125Raise once more, give cis-platinum once more, the endoxan scheme chemotherapy is still effective.
Example 3: patient 49 years old, the both ovaries serous papillary adenocarcinoma IV phase, the left clavicle superior gluteal lymph node biopsy positive, row cis-platinum 1 course of treatment, AC are performed the operation after the chemotherapy in advance, the maximum residual focus 2cm of postoperative, cancerous tissue is commissioned to train fosterly so that tryptic digestion method row is former, and trypsinase concentration is 0.25%, 37 ℃ digested 15 minutes down, digest more complete.The detection telomerase activation is 2.879U, add with detecting telomerase activation behind cis-platinum, the Zorubicin and be respectively 2.133U, 2.215U, telomerase activation descends 25.91%, 23.06% respectively before and after the medication, point out this patient all insensitive to cis-platinum, Zorubicin, and the inhibition rate of tumor cell of mtt assay mensuration cis-platinum, Zorubicin is respectively 13.33%, 10.33%, also point out tumour all insensitive to cis-platinum, Zorubicin, clinical follow up results shows, after the 3rd course of treatment cis-platinum, AC chemotherapy, patient's blood CA
125Do not descend and significantly increase on the contrary, the patient is to cis-platinum, Zorubicin resistance in prompting.
Example 4: patient 54 years old, the left ovarian serous gland cancer III phase, cell subtracts the maximum residual focus of the postoperative that goes out less than 2cm, and cancerous tissue is commissioned to train fosterly so that tryptic digestion method row is former, and trypsinase concentration is 0.125%, 37 ℃ of digestion 15 minutes down, digests more complete.The detection telomerase activation is 1.776U, add with detecting telomerase activation behind cis-platinum, the Zorubicin and be respectively 0.998U, 0.734U, telomerase activation descends 43.81%, 58.67% respectively before and after the medication, point out this patient all responsive to cis-platinum, Zorubicin, and the inhibition rate of tumor cell of mtt assay mensuration cis-platinum, Zorubicin is respectively 40.68%, 87.33%, also point out tumour all responsive to cis-platinum, Zorubicin, clinical follow up results shows that the patient is effective to cis-platinum, AC scheme chemotherapy.Patient's blood CA
125Level continues to descend, and reduces to after the chemotherapy normally to the 4th course of treatment.
Claims (4)
1 one kinds of methods with telomerase activation judgement epithelial ovarian cancer drug susceptibility is characterized in that finishing according to the following steps:
1) get patient's ovarian epithelial cancerous tissue carry out former be commissioned to train foster;
2) get former being commissioned to train and support the original telomerase activation of back tumour cell extracting solution mensuration tumour cell;
3) get the former foster back tumour cell of being commissioned to train and add cis-platinum or Zorubicin continuation cultivation, with the adherent tumour cell of tryptic digestion, tumour cell telomerase activation after the mensuration medication;
4) calculate the decline degree of the original telomerase activation of Telomerase specific activity after the medication, be judged as medicaments insensitive when telomerase activation descends when surpassing 30%, it is insensitive to be lower than at 30% o'clock and to be judged as medicine.
2 in accordance with the method for claim 1, the method that it is characterized in that the epithelial ovarian cancer tissue in primary culture is: the cancerous tissue that will downcut in will performing the operation is put into the sterile culture bottle that contains the RPMI1640 substratum, in super clean bench, open and put into sterile petri dish, with scissors tissue is shredded after washing down blood repeatedly with physiological saline, and wash repeatedly with RPMI1640; Digested 10～20 minutes down at 37 ℃ with 0.125%～0.25% trypsinase then, make single cell suspension to blow and beat repeatedly after the foetal calf serum deactivation, 1500bpm is centrifugal, abandons supernatant liquor, add 10% foetal calf serum with the cell precipitation spin-up with RPMI1640, it is 1 * 10 that cell concn is adjusted in the back
6/ ml is inoculated in the culture dish, at 37 ℃, 5%CO
2Cultivate in the incubator.
3 according to claim 1 or 2 described methods, and it is characterized in that using the TRAP-PCR-ELISA method and detect the preceding telomerase activation of medication, be the ovarian epithelial cancerous tissue to be made single cell suspension with the tryptic digestion method, adjusting tumour cell concentration is 1 * 10
6/ ml gets 2ml and measures original telomerase activation.
4 according to claim 1 or 2 described methods, and it is characterized in that using the TRAP-PCR-ELISA method and detect telomerase activation after the medication, be the ovarian epithelial cancerous tissue to be made single cell suspension with the tryptic digestion method, adjusting tumour cell concentration is 1 * 10
6/ ml gets 2ml and is inoculated in the culture dish, continues to cultivate 12 hours, adds cis-platinum 10 μ g/ml or Zorubicin 1 μ g/ml, continues to cultivate to stop in 72 hours, with the adherent tumour cell of 0.25% tryptic digestion, measures telomerase activation.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101313219B (en) * | 2005-04-01 | 2016-10-05 | 麦德维特科学控股有限公司 | A kind of diagnosis and Therapeutic Method and the reagent that used thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101313219B (en) * | 2005-04-01 | 2016-10-05 | 麦德维特科学控股有限公司 | A kind of diagnosis and Therapeutic Method and the reagent that used thereof |
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