CN106596941A - Detection method for detecting prostate cancer circulating tumor cells - Google Patents
Detection method for detecting prostate cancer circulating tumor cells Download PDFInfo
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- CN106596941A CN106596941A CN201611189125.9A CN201611189125A CN106596941A CN 106596941 A CN106596941 A CN 106596941A CN 201611189125 A CN201611189125 A CN 201611189125A CN 106596941 A CN106596941 A CN 106596941A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
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Abstract
The invention discloses a detection method for detecting prostate cancer circulating tumor cells, wherein the detection method comprises the following specific steps: step one, collection of a peripheral blood sample; step two, blood cell separation; step three, antibody staining; step four, cell immobilization; step five, ethanol dehydration; step six, probe incubation; step seven, cleaning; and step eight, sheet sealing. The method can be used for early detection of prostate cancer, so as to help diagnosis and treatment; the method can specifically detect free tumor cells in a circulating blood system, calculates out the positive rate, and guides doctors to select a treatment scheme according to the positive rate and the cell number condition of patients at different time nodes of a treatment course; the method can perform subsequent detection work, such as single cell sequencing, targeted drug screening and the like, detects other tumor substances simultaneously, is more comprehensive in detection of tumor metastasis, drug resistance, differentiation and other conditions, and has good use effect.
Description
Technical field
The present invention relates to peripheral blood tumor cell detection field, specifically a kind of detection carcinoma of prostate circulating tumor cell
Detection method.
Background technology
Carcinoma of prostate is one of modal malignant tumor of current male, and its sickness rate worldwide occupies pernicious swollen
The 4th of tumor morbidity, occupies Incidence the 6th and sickness rate is in ascendant trend year by year in China.China in 2012
Tumour registration area prostate-cancer incidence is 9.92/10 ten thousand.Carcinoma of prostate age of onset before 55 years old be in reduced levels, 55
Gradually rise after year, sickness rate increases with advancing age, and the peak age is 70～80 years old, becomes serious harm male's body
The important killer of body health.
Circulating tumor cell（CTC）Refer to it is spontaneous or because operation of diagnosis and treatment discharged into by solid tumor primary tumor or metastasis it is outer
The tumor cell of all blood circulations.Metastasis are patients with prostate cancer main causes of death, and CTC is considered neoplasm metastasis
, there is very big application in terms of discovery, the tumor prognosis judgement and individualized treatment in new Tumor biomarkers in " seed "
Potentiality, are one of focuses of domestic and international tumor research.The method of existing capture and enrichment CTC mainly has based on the physics of CTC
Character is captured, such as density, molecular size physical behavior, or based on CTC surface biologicals mark such as EpCAM（Epithelium is thin
Born of the same parents' adhesion molecule）Etc. being enriched with, the Cellsearch detection platform that such as Jing FDA approvals are used, Cellsearch platforms are profits
It is enriched with EpCAM is positive to CTC, is then identified with CK18 fluorescence stainings.But based on physical propertys pair such as density, molecular sizes
CTC is captured, and is easily caused CTC leakage sieves, obtains false negative result.And due to the heterogeneity of different tumors, EpCAM is also more next
More is proved might not there is good expression effect in the tumor cell surface of carcinoma of prostate.Therefore a kind of tool is needed
There is the carcinoma of prostate peripheral circulation tumor cell capture technique of more high specific and sensitivity.
In recent years, a kind of CTC methods of combination feminine gender beneficiation technologies and immunofluorescence hybridization in situ technique are applied to CTC
Detection, the method do not rely on the expression of cell surface EpCAM molecules, and the probe for how selecting specificity recognizes carcinoma of prostate
CTC is the important technology difficult point of this technology, prostate specific membrane antigen（PSMA）It is class specificity in prostata tissue
The antigen molecule of expression, because the important target that it is carcinoma of prostate diagnostic imaging and treatment is increasingly closed in recent years by researcher
Note.Researcher finds that PSMA has expression, and expression intensity in hyperplasia of prostate tissue, normal structure, the cancerous tissue of prostate
Rise successively, in expression in prostate cancer intensity highest；Express in Metastatic Lymph Nodes, Bone tumour stove in carcinoma of prostate
Also it is positive.With going deep into for research, result of study all shows that PSMA is expected to become the prostate of high specific and hypersensitivity
The tumor markerses of disease surveillance, but the detection method not yet having had at present.
The content of the invention
It is an object of the invention to provide a kind of detection method of detection carcinoma of prostate circulating tumor cell, above-mentioned to solve
The problem proposed in background technology.
For achieving the above object, the present invention provides following technical scheme：
A kind of detection method of detection carcinoma of prostate circulating tumor cell, comprises the following steps that：
Step one, gathers peripheral blood sample：A blood taking tube is taken, the periphery whole blood of patients with prostate cancer is extracted, such as need to be sent
Sample is detected in room temperature and in 48 hours；
Step 2, hemocyte is separated：To be placed in horizontal centrifuge after blood taking tube trim, centrifuge is centrifuged at normal temperatures 5-8 point
Clock, then carefully draws supernatant to cellular layer；To in blood taking tube plus cell cleanout fluid and mixing of turning upside down, separately take one from
Heart pipe and add separation of lymphocytes medium, the cell after mixing is carefully transferred to into separation of lymphocytes medium upper strata, then add
Cell cleanout fluid, is added in upper strata after rinse, repeat rinse once；Centrifuge tube is placed in centrifuge, centrifuge is in normal temperature state
Lower centrifugation 4-8 minutes, be divided into three layers after centrifugation, bottom it is red for erythrocyte, the intermediate layer of middle whitish is mainly white
Cell and CTC, upper strata is the blood plasma of yellow, draws whole liquid more than erythrocyte, removes red blood cell layer；Magnetic bead mixing is equal
It is even, dropwise add magnetic bead, horizontal shaker to shake 15-22 minutes at normal temperatures；A centrifuge tube, plus lymph separating medium are taken, will be contained
The suspension for having magnetic bead is transferred to separating medium upper strata, and centrifuge tube is placed in centrifuge, and centrifuge is centrifuged at normal temperatures 5-10
Minute, then bottom separating medium and above liquid are drawn, adsorb 2-3 minutes with big magnetic frame；
Step 3, antibody staining：Antibody is prepared in antibody diluent, the antibody of CD45-Alexa 594 and PSMA-Alexa 488
Antibody concentration is 1mg/ml, and after respectively the ask for antibody of CD45-Alexa 594 and the μ l of 488 antibody of PSMA-Alexa 1 antibody is added
The μ l of diluent 198, then antibody in the cell in step 2 plus after the dilution of 200 μ L, lucifuge room temperature incubation 20 minutes, incubate
Add cell cleanout fluid to 14 mL, centrifuge 2-5 minutes after educating, remove supernatant to 100 μ l；
Step 4, cell is fixed：Cell is with fixative according to 1:1 ratio mixing, piping and druming mixes cell, is evenly coated in microscope slide
On square frame in, the calm air dried overnight in 28-34 degree Celsius of baking oven；
Step 5, ethanol dehydration：Slide is inserted into 1-4 minutes in 100% ethanol decolorization cylinder, the second at the slide back side and edge is wiped
Alcohol is remained, and is dried up with the cold wind of hair-dryer；
Step 6, probe incubation：Plus the CEP8 probes of 10ul, covered and in surrounding with mounting glue mounting, first in 72-
80 degrees Celsius of lower prehybridization 8-12 minutes；Then 15-18 hours are hybridized under 35-40 degree Celsius；
Step 7, cleaning：Tear mounting glue off, slide is inserted into and is rushed in the color jar of liquid liquid, in color jar equipped with probe hybridization
In rock 1-2 minutes, tweezers translation push coverslip, in probe hybridization buffer clean 1-2 time, every time 5 minutes；Then exist
Clean 2-4 time, every time 5 minutes in probe cleanout fluid；
Step 8, mounting：Mounting, covered are carried out using mountant.
As further scheme of the invention：Mountant is the mixture of 4', 6- diamidinos -2-phenylindone and glycerol.
As further scheme of the invention：Magnetic bead adopts CD45 immunomagnetic beadses.
Compared with prior art, the invention has the beneficial effects as follows：The present invention can carry out early detection to carcinoma of prostate, from
And assisted diagnosis and treatment；The present invention can specifically detect the Iisolated tumor cells in blood circulation system, count sun
Property rate, by the positive rate and cell number situation of patient course for the treatment of different time node instruct doctor select therapeutic scheme；
The present invention can carry out subsequent detection work, and such as unicellular sequencing, targeted drug screening can simultaneously detect other tumor standards
Situations such as thing, the more transfer of complete detection tumor, drug resistance and differentiation, using effect is good.
Specific embodiment
The technical scheme of this patent is described in more detail with reference to specific embodiment.
Embodiment 1
LNCaP Human Prostate Cancer Cells are used according to the method described above antibody staining, the antibody prepare in antibody diluent,
The antibody of CD45-Alexa 594 and the antibody concentration of PSMA-Alexa 488 are 1mg/ml, and antibody dilution is added after the 1 μ l that respectively ask for
The μ l of liquid 198.Carry out artificial read tablet at normal temperatures, device therefor includes low speed large capacity centrifuge, mini desktop vacuum pump, miniature
Horizontal decolorization swinging table, trim balance, electrically heated drying cabinet, digital display thermostat water bath, the big magnetic frames of 50 ml, manual single track are adjustable
Pipettor, miniature Blowing drum, Large Copacity electric pipettor, micro centrifuge, vortex mixed instrument, in situ hybridization instrument and fluorescence microscopy
Mirror, the microscopical free scanning software of Nikon of software and using NIS-Elements F 4.00.00 take a picture.
The basis for estimation of CTC most criticals is that CD45 is negative and the non-diploid of No. 8 chromosome numbers.CD45：It is red
CTC visible pale red backgrounds sometimes under passage, clearest differences is that with leukocyte and do not surround nuclear distinct red light
Ring（Sometimes the ring of light is imperfect）.
CEP8：Monomer, diploid or many bodies, signal is apparent under red or orange passage.Tumor mark passage：Green letter
Number, different according to tumor mark expression, signal light and shade differs, and some tumor cells are not expressed.DAPI：Blueness, circle or oval
CTC judges：
DAPI+/CD 45-/tumor mark+/ No. 8 chromosomes+（Arbitrary number chromosome）.
DAPI+/CD 45-/tumor mark -/No. 8 chromosomes+（1 body, 3 bodies, 4 bodies, >=5 bodies）, due to CTC
Some tumor rotating savings degradeds during interstitial, so this CTC is relatively conventional, additionally, the CTC in peripheral blood can be divided into list
Individual CTC and cancer embolus（CTM, >=2 cancerous cell）, cancer embolus cell plays particularly critical in neoplasm metastasis and relapsing course
Effect, for such cell can not be ignored.
Embodiment 2
Peripheral blood in patients CTC testing results and related history, are exemplified below：Patient, man, 73 years old.Patient is before 5 years because of carcinoma of prostate
Row Prostate Cancer after Radical, because prostatic cavity wall local recurrence photon radiation is treated after 2 years, accumulated dose reaches 68Gy/34Fx, radiotherapy
Give endocrine therapy and chemotherapy in succession afterwards, but PSA progressive is raised, 2016.2.26 row SPECT（99mTc-PSMA）Whole body is examined
Look into, point out：Carcinoma of prostate is postoperative, and multiple lymphadenectasis by left iliac vessels, after peritoneum, radioactive uptake increases, it is considered to shift；
2016.3.3 row is based on prostate specific membrane antigen（PSMA）Negative concentration method for molecular marked compound detects Peripheral Circulation
Tumor cell, as a result shows：In 7.5ml peripheral bloods, detect CTC and amount to 15, the CTC of wherein PSMA+ 3（Respectively
3 bodies+, 4 bodies+, 5 bodies+）, Detection results are authentic and valid.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of spirit or essential attributes without departing substantially from the present invention, the present invention can be in other specific forms realized.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit is required rather than described above is limited, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.
Moreover, it will be appreciated that although this specification is been described by according to embodiment, not each embodiment is only wrapped
Containing an independent technical scheme, this narrating mode of description is only that for clarity those skilled in the art should
Using description as an entirety, the technical scheme in each embodiment can also Jing it is appropriately combined, form those skilled in the art
Understandable other embodiment.
Claims (3)
1. it is a kind of detection carcinoma of prostate circulating tumor cell detection method, it is characterised in that comprise the following steps that：
Step one, gathers peripheral blood sample：A blood taking tube is taken, the periphery whole blood of patients with prostate cancer is extracted, such as need to be sent
Sample is detected in room temperature and in 48 hours；
Step 2, hemocyte is separated：To be placed in horizontal centrifuge after blood taking tube trim, centrifuge is centrifuged at normal temperatures 5-8 point
Clock, then carefully draws supernatant to cellular layer；To in blood taking tube plus cell cleanout fluid and mixing of turning upside down, separately take one from
Heart pipe and add separation of lymphocytes medium, the cell after mixing is carefully transferred to into separation of lymphocytes medium upper strata, then add
Cell cleanout fluid, is added in upper strata after rinse, repeat rinse once；Centrifuge tube is placed in centrifuge, centrifuge is in normal temperature state
Lower centrifugation 4-8 minutes, be divided into three layers after centrifugation, bottom it is red for erythrocyte, the intermediate layer of middle whitish is mainly white
Cell and CTC, upper strata is the blood plasma of yellow, draws whole liquid more than erythrocyte, removes red blood cell layer；Magnetic bead mixing is equal
It is even, dropwise add magnetic bead, horizontal shaker to shake 15-22 minutes at normal temperatures；A centrifuge tube, plus lymph separating medium are taken, will be contained
The suspension for having magnetic bead is transferred to separating medium upper strata, and centrifuge tube is placed in centrifuge, and centrifuge is centrifuged at normal temperatures 5-10
Minute, then bottom separating medium and above liquid are drawn, adsorb 2-3 minutes with big magnetic frame；
Step 3, antibody staining：Antibody is prepared in antibody diluent, the antibody of CD45-Alexa 594 and PSMA-Alexa 488
Antibody concentration is 1mg/ml, and after respectively the ask for antibody of CD45-Alexa 594 and the μ l of 488 antibody of PSMA-Alexa 1 antibody is added
The μ l of diluent 198, then antibody in the cell in step 2 plus after the dilution of 200 μ L, lucifuge room temperature incubation 20 minutes, incubate
Add cell cleanout fluid to 14 mL, centrifuge 2-5 minutes after educating, remove supernatant to 100 μ l；
Step 4, cell is fixed：Cell is with fixative according to 1:1 ratio mixing, piping and druming mixes cell, is evenly coated in microscope slide
On square frame in, the calm air dried overnight in 28-34 degree Celsius of baking oven；
Step 5, ethanol dehydration：Slide is inserted into 1-4 minutes in 100% ethanol decolorization cylinder, the second at the slide back side and edge is wiped
Alcohol is remained, and is dried up with the cold wind of hair-dryer；
Step 6, probe incubation：Plus the CEP8 probes of 10ul, covered and in surrounding with mounting glue mounting, first in 72-
80 degrees Celsius of lower prehybridization 8-12 minutes；Then 15-18 hours are hybridized under 35-40 degree Celsius；
Step 7, cleaning：Tear mounting glue off, slide is inserted into and is rushed in the color jar of liquid liquid, in color jar equipped with probe hybridization
In rock 1-2 minutes, tweezers translation push coverslip, in probe hybridization buffer clean 1-2 time, every time 5 minutes；Then exist
Clean 2-4 time, every time 5 minutes in probe cleanout fluid；
Step 8, mounting：Mounting, covered are carried out using mountant.
2. it is according to claim 1 detection carcinoma of prostate circulating tumor cell detection method, it is characterised in that the envelope
Tablet is the mixture of 4', 6- diamidinos -2-phenylindone and glycerol.
3. it is according to claim 1 and 2 detection carcinoma of prostate circulating tumor cell detection method, it is characterised in that institute
Magnetic bead is stated using CD45 immunomagnetic beadses.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107748256A (en) * | 2017-10-11 | 2018-03-02 | 上海医盈网络科技有限公司 | A kind of liquid biopsy detection method of circulating tumor cell |
| CN109557296A (en) * | 2018-11-22 | 2019-04-02 | 珠海澳加动力生物科技有限公司 | A kind of method of cycle detection tumour cell drug susceptibility |
| CN109991205A (en) * | 2019-05-05 | 2019-07-09 | 中国科学院重庆绿色智能技术研究院 | A kind of counting algorithm of circulating tumor cell and application |
| CN115201476A (en) * | 2022-09-15 | 2022-10-18 | 长沙普方德医疗器械有限公司 | Circulating tumor cell detection equipment and use method thereof |
-
2016
- 2016-12-21 CN CN201611189125.9A patent/CN106596941A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107748256A (en) * | 2017-10-11 | 2018-03-02 | 上海医盈网络科技有限公司 | A kind of liquid biopsy detection method of circulating tumor cell |
| CN107748256B (en) * | 2017-10-11 | 2020-04-14 | 厦门骁科码生物科技有限公司 | Liquid biopsy detection method for circulating tumor cells |
| CN109557296A (en) * | 2018-11-22 | 2019-04-02 | 珠海澳加动力生物科技有限公司 | A kind of method of cycle detection tumour cell drug susceptibility |
| CN109557296B (en) * | 2018-11-22 | 2022-05-20 | 珠海澳加动力生物科技有限公司 | Method for circularly detecting drug sensitivity of tumor cells |
| CN109991205A (en) * | 2019-05-05 | 2019-07-09 | 中国科学院重庆绿色智能技术研究院 | A kind of counting algorithm of circulating tumor cell and application |
| CN115201476A (en) * | 2022-09-15 | 2022-10-18 | 长沙普方德医疗器械有限公司 | Circulating tumor cell detection equipment and use method thereof |
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