CN106645724A - Method for detecting CTC (circulating tumor cell) surface marker molecule GPC3 (glypican-3) - Google Patents
Method for detecting CTC (circulating tumor cell) surface marker molecule GPC3 (glypican-3) Download PDFInfo
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- CN106645724A CN106645724A CN201611189879.4A CN201611189879A CN106645724A CN 106645724 A CN106645724 A CN 106645724A CN 201611189879 A CN201611189879 A CN 201611189879A CN 106645724 A CN106645724 A CN 106645724A
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- G—PHYSICS
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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
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
- G01N33/531—Production of immunochemical test materials
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- G01N33/533—Production of labelled immunochemicals with fluorescent label
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Abstract
The invention belongs to the field of molecular biology, and particularly discloses a method for detecting a CTC (circulating tumor cell) surface marker molecule GPC3 (glypican-3). The method comprises the following steps: performing erythrocyte splitting on blood, spreading and enriching all of left karyotes (mainly comprising CTCs and lymphocytes) on a nano-substrate for fixation by virtue of a nanotechnology, marking all the cells by virtue of a nucleus fluorescent dye DAPI, performing positive selection by virtue of a tumor immune fluorescent marker, i.e. a cytokeratin antibody anti-CK, and since a CTC surface has a specific expression of the GPC3, incubating all the cells by virtue of a GPC3 primary antibody, performing incubation by virtue of a secondary antibody marked with an FITC fluorescence group, and finally performing scanning by virtue of a high-throughput technology. Compared with the prior art, the detection method has the advantages of convenience, rapidness, economy, high sensitivity and specificity and capability of overcoming the defects of large sample amount requirement and incomplete reaction of GPC3 detection in the prior art.
Description
[technical field]
The invention belongs to biology field, specifically a kind of circulating tumor cell surface markers phosphatidyl
The detection method of inositol proteoglycans -3.
[background technology]
Circulating tumor cell (circulating tumor cell；CTC it is) to shed into peripheral blood from primary tumors
Tumour cell, suitable microenvironment can be selected with blood flow, plant in distal organs, form MET.At present, periphery
Blood CTC detection techniques can only realize qualitative and relative quantification the detection of CTC in human peripheral blood, can not track in blood
Which organ CTC derives from, and CTC is the tumour cell split away off from primary tumor, and its surface is contained special on primary tumor
The marker molecule of opposite sex expression.If glypican-3 (glypican-3, GPC3) is that one kind is aggregated in cell membrane table
The heparin class sulfated proteoglycans in face, can be scheduled on cell surface by glycosyl-phosphatidyl inositol anchor, and the propagation to tumour cell is moved
Move and there is regulating and controlling effect.There are some researches show, GPC3 has a high expression in primary hepatocyte hepatocarcinoma HCC, and cancer beside organism,
Cirrhosis, hepatitis tissue, adult normal hepatic tissue low expression are not expressed.Therefore, it is thin if a kind of detection circulating tumor can be provided
The method of cellular surface GPC3, will have very important significance.
[content of the invention]
Present invention aim to solve above-mentioned deficiency and provide a kind of circulating tumor cell surface markers phosphorus
The detection method of acyl inositol proteoglycans -3, the detection method is easy, quick, economical, and sensitivity is high, and specificity is good, can
Solving prior art detection GPC3 needs the halfway deficiency of the big reaction of specimen amount.
A kind of circulating tumor cell surface markers Monophosphoinositideproteoglycans proteoglycans-3 is designed for achieving the above object
Detection method, comprises the following steps：
1) whole blood is processed：A. erythrocyte cracked liquid, room temperature is added to place 15min in whole blood, period uniformly rocks；B. in
200RCF is centrifuged 5 minutes, absorbs supernatant, retains cell；C. 1%FPBS is added in centrifuge tube, is mixed and is centrifuged 5 after 200RCF
Minute, remove supernatant；
2) cell is enriched with entirely：A. add FPBS to proceed in the culture dish handled well after mixing, culture dish is placed in into 37 DEG C and is shaken
45min is cultivated in bed；B. culture dish is placed in 4 DEG C of refrigerators after cultivating, stands 1min；
3) cell is fixed：A. FPBS is absorbed, is added in culture dish and 4 DEG C of standing 1min is placed in after 4% formaldehyde；B. first is absorbed
Aldehyde, adds 1mL methyl alcohol, is placed in -20 DEG C of 20min；C. methyl alcohol is absorbed again, every time with 2mL PBSs three times；
4) closing and antibody incubation：A. skimmed milk powers of the 5%m/V containing 0.02%Tween-20 is added in culture dish, is taken off
Fat milk powder is configured with PBS, and 4 DEG C of lucifuges are incubated 1h；B. absorb after skimmed milk power, add PBST to clean 4 times along wall, each 5min；
C. add anti-CK and GPC3 mono- to resist in confining liquid, add in culture dish along wall after being well mixed, 4 DEG C of lucifuge overnight incubations；
D. mixed liquor is absorbed, adds GPC3 bis- to resist in confining liquid, added in culture dish along wall after mixing, 4 DEG C of lucifuges are incubated 1h；E. inhale
Except antibody, cleaned with PBST three times, add DAPI, normal temperature lucifuge incubation 30min；
5) scan：Antibody is absorbed, is cleaned with PBST three times, add PBS scannings.
Further, the FPBS is formed by FBS, PBS configuration, and its volume ratio is FBS:PBS=1:99.
Further, step 4) in, the PBS containing 0.02%Tween-20 in PBST.
Further, step 5) in, using high flux polychrome imaging analysis, the optical filter of CY5, FITC and DAPI is selected,
Passage surface fluorescence color is observed, shows blue then DAPI+, do not show blue then DAPI-, aobvious green then GPC3+, do not show green then
GPC3-, aobvious red then CK+, does not show red then CK-, according to fluorescence developing, by DAPI+ and the point of CK+ is considered CTC, if should
There is GPC3+ on CTC, then the CTC derives from liver.
The present invention compared with the existing technology, first combines CTC detections and GPC3 marker detections, by blood
Erythrocyte splitting is carried out, all tiling is enriched in make remaining karyocyte (mainly CTC and lymphocyte) using nanometer technology
It is fixed in nanometer substrate, then all cells are marked with nucleus fluorescent dye DAPI, afterwards by tumour immunity fluorescence mark
Will thing anti-cytokeratin Ab anti-CK carries out positive-selecting, and because CTC surfaces have, GPC3's is specific expressed, uses GPC3
One all cells of anti-incubation, then use be marked with FITC fluorophors two anti-incubations, finally by high-throughput techniques scanning, finally
Detection to circulating tumor cell surface markers Monophosphoinositideproteoglycans proteoglycans-3 is realized, the detection method is easy, quick,
Economy, sensitivity is high, and specificity is good, and solving prior art detection GPC3 needs the halfway deficiency of the big reaction of specimen amount.
[specific embodiment]
Further explained below is made to the present invention with reference to specific embodiment：
The invention provides a kind of detection side of circulating tumor cell surface markers Monophosphoinositideproteoglycans proteoglycans-3
Method, specifically includes following steps：
1) whole blood is processed：
A. 200 μ 1 × erythrocyte cracked liquids of L, room temperature are added to place 15min in 2mL whole bloods, period uniformly rocks.
B.200RCF it is centrifuged 5 minutes, absorbs supernatant, retains cell.
C. 2mL 1%FPBS (V (FBS) are added in centrifuge tube:V (PBS)=1:99), mix and be centrifuged 5 after 200RCF
Minute, remove supernatant.
2) cell is enriched with entirely：
A. add 1mL FPBS to proceed in the culture dish handled well after mixing, culture dish is placed in 37 DEG C of shaking tables and is cultivated
45min。
B. culture dish is placed in 4 DEG C of refrigerators after cultivating, stands 1min.
3) cell is fixed：
A. FPBS is absorbed, is added in culture dish and 4 DEG C of standing 1min is placed in after 4% formaldehyde.
B. formaldehyde is absorbed, 1mL methyl alcohol is added, -20 DEG C of 20min are placed in.
C. methyl alcohol is absorbed, every time with 2mL PBSs three times.(note：When adding liquid every time, add along culture dish wall, keep away
Exempt to rush in cell.
4) closing and antibody incubation：
A. the skimmed milk power (being configured with PBS) for adding 1mL 5% (m/V) to contain 0.02%Tween-20 in culture dish, 4 DEG C
Lucifuge is incubated 1h.
B. absorb after skimmed milk power, add 2mL PBST (PBS containing 0.02%Tween-20) cleaning 4 times along wall, every time
5min。
C. the 1 μ L anti-CK and anti-(proteintech of 2 μ LGPC3 mono- are added in 500 μ L confining liquids；25175-1-
AP), add in culture dish along wall after being well mixed, 4 DEG C of lucifuge overnight incubations.
D. mixed liquor is absorbed, anti-(the three arrows biologies of 5 μ L GPC3 bis- is added in 500 μ L confining liquids；GR200G-02C), mix
Add in culture dish along wall after even, 4 DEG C of lucifuges are incubated 1h.
E. antibody is absorbed, is cleaned with PBST three times, add 1mL nucleus dyestuff DAPI (4,6- connection narrow -2-phenylindone),
Normal temperature lucifuge is incubated 30min.
5) scan：Antibody is absorbed, is cleaned with 2mL PBST three times, add 1mL PBS scannings.
Above-mentioned steps 5) in, using high flux polychrome imaging analysis, the optical filter of CY5, FITC and DAPI being selected, observation is logical
Road surface fluorescence color, shows blue then DAPI+, does not show blue then DAPI-, aobvious green then GPC3+, does not show green then GPC3-,
Aobvious red then CK+, does not show red then CK-, according to fluorescence developing, by DAPI+ and " point " of CK+ is considered CTC, if the CTC
On there is GPC3+, then the CTC derives from liver, otherwise, is not then, and the detection method is easy, quick, economical, sensitivity
Height, specificity is good.
The present invention is not limited by above-mentioned embodiment, other any Spirit Essences and principle without departing from the present invention
Lower made change, modification, replacement, combination, simplification, should be equivalent substitute mode, be included in the protection model of the present invention
Within enclosing.
Claims (4)
1. a kind of detection method of circulating tumor cell surface markers Monophosphoinositideproteoglycans proteoglycans-3, it is characterised in that
Comprise the following steps：
1) whole blood is processed：
A. erythrocyte cracked liquid, room temperature is added to place 15min in whole blood, period uniformly rocks；
B. it is centrifuged 5 minutes in 200RCF, absorbs supernatant, retains cell；
C. 1%FPBS is added in centrifuge tube, is mixed and is centrifuged 5 minutes after 200RCF, remove supernatant；
2) cell is enriched with entirely：
A. add FPBS to proceed in the culture dish handled well after mixing, culture dish is placed in 37 DEG C of shaking tables and cultivates 45min；
B. culture dish is placed in 4 DEG C of refrigerators after cultivating, stands 1min；
3) cell is fixed：
A. FPBS is absorbed, is added in culture dish and 4 DEG C of standing 1min is placed in after 4% formaldehyde；
B. formaldehyde is absorbed, 1mL methyl alcohol is added, -20 DEG C of 20min are placed in；
C. methyl alcohol is absorbed again, every time with 2mL PBSs three times；
4) closing and antibody incubation：
A. skimmed milk powers of the 5%m/V containing 0.02%Tween-20, skimmed milk power is added to be configured with PBS in culture dish, 4 DEG C are kept away
Light is incubated 1h；
B. absorb after skimmed milk power, add PBST to clean 4 times along wall, each 5min；
C. add anti-CK and GPC3 mono- to resist in confining liquid, add in culture dish along wall after being well mixed, 4 DEG C of lucifuge incubations
Overnight；
D. mixed liquor is absorbed, adds GPC3 bis- to resist in confining liquid, added in culture dish along wall after mixing, 4 DEG C of lucifuges are incubated 1h；
E. antibody is absorbed, is cleaned with PBST three times, add DAPI, normal temperature lucifuge incubation 30min；
5) scan：
Antibody is absorbed, is cleaned with PBST three times, add PBS scannings.
2. the method for claim 1, it is characterised in that：The FPBS is formed by FBS, PBS configuration, and its volume ratio is
FBS:PBS=1:99.
3. the method for claim 1, it is characterised in that：Step 4) in, the PBS containing 0.02%Tween-20 in PBST.
4. the method for claim 1, it is characterised in that：Step 5) in, using high flux polychrome imaging analysis, select
The optical filter of CY5, FITC and DAPI, observes passage surface fluorescence color, shows blue then DAPI+, does not show blue then DAPI-,
Aobvious green then GPC3+, does not show green then GPC3-, aobvious red then CK+, does not show redness then CK-, according to fluorescence developing, by DAPI+
And the point of CK+ is considered CTC, if there is GPC3+ on the CTC, then the CTC derives from liver.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108507992A (en) * | 2018-04-09 | 2018-09-07 | 苏州大学附属第医院 | The detection method of circulating tumor cell surface markers PD-L1 |
| CN109541214A (en) * | 2018-12-13 | 2019-03-29 | 钱军 | The detection method of circulating tumor cell surface markers HER2 |
| CN112029728A (en) * | 2020-09-18 | 2020-12-04 | 山东大学 | Fluorescent magnetic nano-composite and preparation method and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102043047A (en) * | 2010-11-02 | 2011-05-04 | 中国科学院武汉病毒研究所 | Rapid and economical immunofluorescence method based on Fisher |
| CN104062428A (en) * | 2014-07-09 | 2014-09-24 | 郑勤 | Kit for detecting circulating tumor cells |
| CN104807996A (en) * | 2014-01-27 | 2015-07-29 | 中国医学科学院肿瘤医院 | Uses of cell surface marker molecule in detection of liver cancer circulating tumor cells |
| CN105067808A (en) * | 2015-07-30 | 2015-11-18 | 南京医科大学 | High-purity circulation tumor cell enriching method and kit |
| CN105115878A (en) * | 2015-09-11 | 2015-12-02 | 上海交通大学 | Circulating tumor cell detection kit, preparing method thereof and application thereof |
| CN105277697A (en) * | 2015-10-27 | 2016-01-27 | 上海芯超生物科技有限公司 | Method for measuring number of circulating epithelial tumor cells in blood |
| CN105717104A (en) * | 2016-01-28 | 2016-06-29 | 山东省肿瘤防治研究院 | Peripheral blood GPC3 detection method for hepatocellular carcinoma patient |
-
2016
- 2016-12-21 CN CN201611189879.4A patent/CN106645724A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102043047A (en) * | 2010-11-02 | 2011-05-04 | 中国科学院武汉病毒研究所 | Rapid and economical immunofluorescence method based on Fisher |
| CN104807996A (en) * | 2014-01-27 | 2015-07-29 | 中国医学科学院肿瘤医院 | Uses of cell surface marker molecule in detection of liver cancer circulating tumor cells |
| CN104062428A (en) * | 2014-07-09 | 2014-09-24 | 郑勤 | Kit for detecting circulating tumor cells |
| CN105067808A (en) * | 2015-07-30 | 2015-11-18 | 南京医科大学 | High-purity circulation tumor cell enriching method and kit |
| CN105115878A (en) * | 2015-09-11 | 2015-12-02 | 上海交通大学 | Circulating tumor cell detection kit, preparing method thereof and application thereof |
| CN105277697A (en) * | 2015-10-27 | 2016-01-27 | 上海芯超生物科技有限公司 | Method for measuring number of circulating epithelial tumor cells in blood |
| CN105717104A (en) * | 2016-01-28 | 2016-06-29 | 山东省肿瘤防治研究院 | Peripheral blood GPC3 detection method for hepatocellular carcinoma patient |
Non-Patent Citations (1)
| Title |
|---|
| 吴明健: "抗-GPC3抗体的制备及肝癌循环肿瘤细胞检测方法研究", 《中国优秀博硕士学位论文全文数据库(硕士) 医药卫生科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108507992A (en) * | 2018-04-09 | 2018-09-07 | 苏州大学附属第医院 | The detection method of circulating tumor cell surface markers PD-L1 |
| CN109541214A (en) * | 2018-12-13 | 2019-03-29 | 钱军 | The detection method of circulating tumor cell surface markers HER2 |
| CN112029728A (en) * | 2020-09-18 | 2020-12-04 | 山东大学 | Fluorescent magnetic nano-composite and preparation method and application thereof |
| CN112029728B (en) * | 2020-09-18 | 2022-07-29 | 山东大学 | Fluorescent magnetic nano-composite and preparation method and application thereof |
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