CN106771185A - A kind of nasopharyngeal carcinoma circulating tumor cell detection kit - Google Patents
A kind of nasopharyngeal carcinoma circulating tumor cell detection kit Download PDFInfo
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- CN106771185A CN106771185A CN201611103862.2A CN201611103862A CN106771185A CN 106771185 A CN106771185 A CN 106771185A CN 201611103862 A CN201611103862 A CN 201611103862A CN 106771185 A CN106771185 A CN 106771185A
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Abstract
The invention provides a kind of nasopharyngeal carcinoma circulating tumor cell detection kit, it is made up of human peripheral leucocytes removal part and immunofluorescence in situ hybridization identification part, wherein human peripheral leucocytes removal part includes dilution buffer, erythrocyte cracked liquid, density gradient centrifugation medium, cell fixer A and immunomagnetic beads, and wherein erythrocyte cracked liquid is mixed by ammonium chloride, sodium acid carbonate and EDETATE SODIUM solution.Described immunofluorescence in situ hybridization identification part includes buffer solution, hybridizes fixer B, the CEP8 probes of biotin labeling, the CD45 antibody of red quantum dot mark, the quantum dot-labeled Streptavidin of green, mountant and bovine serum albumin(BSA) powder.The kit combines immune magnetic enrichment isolation technics, fluorescence in situ hybridization technique, Immunofluorescence technology, nasopharyngeal carcinoma circulating tumor cell counting technology, can accurately determine nasopharyngeal carcinoma circulating tumor cell number in sample.
Description
Technical field
The invention provides a kind of detection kit of nasopharyngeal carcinoma circulating tumor cell, belong to nasopharyngeal carcinoma circulating tumor cell
Detection field.
Background technology
Circulating tumor cell (Circulating Tumor Cells, CTCs) refers to the tumour cell entered into blood.This
Partial tumors cell is participated in the middle of blood circulation, and as sanguimotor direction can be migrated to linked groups or device
Official, develops into tumor focus under suitable conditions.CTCs main sources are divided into two aspects：One to come off, i.e., by primary tumor or
MET splits away off penetrate blood wall and enters into blood.Two is stage entrance earlier, i.e., form image in not yet propagation
When learning visible tumor focus tissue, entered into blood in advance in the form of cancerous tumor cell, therefore theoretically, CTCs's
Occur will be earlier than iconography to tumor entity discovery.
The common inspection method of nasopharyngeal carcinoma has：Magnetic resonant imaging examination, CT examination, pharyngorhinoscopy, X-ray examination, blood
It is clear to learn diagnosis etc..Common being mainly based on serodiagnosis is disliked according to Epstein-Barr virus antibody level in patients with nasopharyngeal carcinoma with other
There is notable difference between tumor patient and Healthy People in property.
Pharynx nasalis anatomical position is hidden, and nasopharyngeal carcinoma early symptom is not true to type, clinically easy delay diagnosis, should especially improve
It is vigilant.And the treatment method of nasopharyngeal carcinoma is mainly based on chemicotherapy, Nasopharyngeal Carcinoma Patients circulating tumor cell is detected and monitored
Quantity can point out early diagnosis, and carry out the assessment of chemicotherapy curative effect.
Detected using people's periphery whole blood, in phase morning, noon and afternoon Nasopharyngeal Carcinoma Patients blood, simply by the presence of entering blood circulation
Nasopharyngeal carcinoma tumor cell, can be detected.Because CTC is few in the quantity of peripheral blood, generally need to be in about 100,000,000 leucocytes
With only several tumour cells are found in 50,000,000,000 red blood cells, therefore in order to improve the recall rate of CTC, generally must be in detection
Before carry out CTC enrichments.The enrichment method of current CTC by its principle be broadly divided into concentration method based on antigen-antibody reaction principle and
Enrichment method based on cellular morphology.And the detection method of CTC is numerous, two major classes can be divided into according to Cleaning Principle：Cytometer
Number method and nucleic acid detection method, the former is mainly including various immunocytochemical techniques, flow cytometry, immunofluorescence technique etc.；
The latter is mainly including polymerase chain reaction, RT-polymerase chain reaction and its various improved technologies etc..Current detection side
Generally existing has following defect in method：Because the leukocyte removal efficiency in peripheral blood is not high, leucocyte can be right as karyocyte
The enrichment of tumour cell is interfered.Additionally, haematogenous abnormal cell also can (circulating tumor be thin to non-haematogenous abnormal cell
Born of the same parents) screening interfere.Additionally, at present mostly using the method capture circulating tumor cell of positive sorting, using magnetic bead surfaces
Coupling has specific antibody (such as Epcam, CK19) to capture tumour cell, and the cell surface that the method is obtained carries magnetic bead, leads
Cause cytoactive reduction.And due to the relation of mark, cause the tumor cell enrichment rate of recovery relatively low.
The content of the invention
The present invention solves deficiency of the prior art, there is provided a kind of nasopharyngeal carcinoma circulating tumor cell detection kit,
The kit combines immune magnetic enrichment isolation technics, fluorescence in situ hybridization technique, Immunofluorescence technology, nasopharyngeal carcinoma and follows
Ring tumour cell counting technology, can accurately determine nasopharyngeal carcinoma circulating tumor cell number in sample.
Realize technical scheme that above-mentioned purpose of the present invention used for：
A kind of nasopharyngeal carcinoma circulating tumor cell detection kit, it is former by human peripheral leucocytes removal part and immunofluorescence
Position hybridization identification part constitutes, and wherein human peripheral leucocytes removal part includes dilution buffer, erythrocyte cracked liquid, density
Gradient centrifugation medium, cell fixer A and immunomagnetic beads, wherein erythrocyte cracked liquid by ammonium chloride, sodium acid carbonate and
EDETATE SODIUM solution is mixed, the magnetic bead of the antibody of the immunomagnetic beads anti-human LCA that has been covalent coupling,
The antibody is CD45, and immunomagnetic beads particle diameter used is 0.2~1.5 μm；Cell fixer A functional components are alcohols；
Described immunofluorescence in situ hybridization identification part includes buffer solution, hybridizes fixer B, the CEP8 of biotin labeling
Probe, the CD45 antibody of red quantum dot mark, the quantum dot-labeled Streptavidin of green, mountant and bovine serum albumin
White powder end；The hybridization fixer B functional components are paraformaldehyde, and the CEP8 probes can be by the side of FISH
Method carries out probe mark to tumour cell, and the CD45 antibody of the red quantum dot mark is dyeed and it to remaining leucocyte
Launch wavelength is 585nm~625nm, and the quantum dot-labeled Streptavidin of green can be combined so as to right with CEP8 probes
DNA fragmentation is marked, and its launch wavelength is 525nm~545nm, and the mountant contains what can be combined with DNA strengths
Fluorescent dye DAPI.
The pH value of described erythrocyte cracked liquid is 7.0~8.0, and wherein the concentration of ammonium chloride is 0.5~2M, sodium acid carbonate
Concentration is 10~200mM, and the concentration of EDETATE SODIUM is 1~10mM.
The CEP8 probes of described biotin labeling are purchased from branch and subsidiaries of Abbott Laboratories of the U.S..
The launch wavelength of the CD45 antibody of red quantum dot mark is 605nm, the quantum dot-labeled Streptavidin of green
Launch wavelength be 525nm, with albumen be attached quantum dot by the way of covalent coupling by both of which.
The density of described density gradient centrifugation medium is 1.02~1.5g/cm3, its active ingredient is sucrose, and sucrose
Mass concentration be 5%~60%.
Compared with prior art, the present invention has advantages below：The present invention is obtained using the method for the negative enrichment of negative sorting
Activity of tumor cells it is high, the tumor cell enrichment rate of recovery be more than 80%.The fluorescence quantum that kit is used is used as non-blood source
Property gene unconventionality type cell marker for determination thing, it is high to be mainly characterized by fluorescent brightness, stable performance, is difficult bleaching, it is easy to observe.
, using the negative method being enriched with, resulting tumor cell surface is complete, and activity is high, more conveniently carries out for the present invention
Follow-up cultivation and molecular marker analyte detection.
The present invention substantially increases circulating tumor thin using blood plasma, erythrocyte splitting technology, density gradient centrifugation is removed
The accumulation rate of born of the same parents, leukocyte removal efficiency is more than 99.99%.
The present invention is using FISH and Immunofluorescence technology to the circulating tumor cell that is enriched to
Identified, No. 8 chromosome probes are marked to DNA of tumor cell sequence, as a result can show triploid, tetraploid, many times
Body signal.It is not only restricted to the expression of epithelium mark using the method for FISH, improves the sensitiveness of detection and special
Property.The CD45 antibody of red quantum dot mark is dyeed to leukocyte surface, is made a distinction with tumour cell.Counted to get
Tumour it is carefully accurate.
CTC beneficiation technologies of the present invention use the negative concentration method based on immune magnetic principle, by using specific Dan Ke
The grand coated immune magnetic particle of antibody (CD45) can remove in blood more than 99.99% leucocyte, farthest reduce the back of the body
The interference of scape karyocyte, so as to reach the purpose of enriched tumor cell.Because not relying on the expression of TCSA,
CTC enrichment methods of the present invention possess certain versatility in each epithelial origin solid tumor, can reach tumour cell higher and return
Yield.To exclude the interference of haematogenous abnormal cell, original creation technology of the present invention：By from specific probe by FISH with it is immune
Cytochemical Technique is combined, and using fluorescence quantum as label, quickly filters out non-haematogenous gene unconventionality type cell
(tumour cell).
Brief description of the drawings
The overhaul flow chart of the nasopharyngeal carcinoma circulating tumor cell detection kit that Fig. 1 is provided for the present invention；
Fig. 2 is the clinical samples detection data analysis chart in the embodiment of the present invention.
Specific embodiment
Detailed specific description is done to the present invention with reference to specific embodiment, but protection scope of the present invention not office
It is limited to following examples.
Nasopharyngeal carcinoma circulating tumor cell detection kit provided by the present invention by human peripheral leucocytes removal part and
Immunofluorescence in situ hybridization identification part constitutes, and wherein human peripheral leucocytes removal part includes dilution buffer, red blood cell
Lysate, density gradient centrifugation medium, cell fixer A and immunomagnetic beads, wherein erythrocyte cracked liquid is by ammonium chloride, carbonic acid
Hydrogen sodium and EDETATE SODIUM solution are mixed, the antibody of the immunomagnetic beads anti-human LCA that has been covalent coupling
Magnetic bead, the antibody be CD45, immunomagnetic beads particle diameter used be 0.2~1.5 μm.The pH value of described erythrocyte cracked liquid is
7.0~8.0, the wherein concentration of ammonium chloride is 0.5~2M, and sodium acid carbonate concentration is 10~200mM, and the concentration of EDETATE SODIUM is 1
~10mM.The density of described density gradient centrifugation medium is 1.02~1.5g/cm3, its active ingredient is sucrose, and sucrose
Mass concentration is 5%~60%.The cell fixer A main components are alcohols.
Described immunofluorescence in situ hybridization identification part includes buffer solution, hybridizes fixer B, the CEP8 of biotin labeling
Probe, the CD45 antibody of red quantum dot mark, the quantum dot-labeled Streptavidin of green, mountant and and cow's serum
Albumin powder, the CEP8 probes can carry out probe mark to tumour cell by the method for FISH, described
Biotin labeling CEP8 probes be purchased from branch and subsidiaries of Abbott Laboratories of the U.S..The hybridization fixer B is to before aging dehydration
Cell is fixed, and main component is paraformaldehyde.The CD45 antibody of the red quantum dot mark is carried out to remaining leucocyte
Dye and its launch wavelength is 585nm~625nm, the launch wavelength of the CD45 antibody of red quantum dot mark is in the present embodiment
605nm, the quantum dot-labeled Streptavidin of green can be combined so as to be marked to DNA fragmentation with CEP8 probes, and
Its launch wavelength is 525nm~545nm, and the launch wavelength of the quantum dot-labeled Streptavidin of the present embodiment Green is
525nm.The CD45 antibody of red quantum dot mark is with green quantum dot-labeled Streptavidin using the side of covalent coupling
With albumen be attached quantum dot by formula.The mountant contains the fluorescent dye DAPI that can be combined with DNA strengths.
Nasopharyngeal carcinoma circulating tumor cell detection kit provided by the present invention overhaul flow chart such as Fig. 1 institutes when in use
Show, specific step is as follows：
1st, blood plasma and red blood cell are removed：5~10min removals are centrifuged after buffer solution 450g~1000g is added in blood sample
Blood plasma, is subsequently adding 450g~1000g erythrocyte cracked liquids and 8-12min is rotated in shaking table, obtains white after 5~10min of centrifugation
Cell and tumour cell.
2nd, leucocyte removal：Immune magnetic particle is added in the leucocyte and tumour cell mixture that will obtain in horizontal shaker
Middle reaction, immune magnetic particle surface coupling has the antibody of anti-human LCA, magnetic particle specificity and leucocyte knot
Close, can be combined with sample 99.99% leucocyte, so as to reach the purpose of removal leucocyte.
3rd, density gradient centrifugation：The magnetic particle and tumour cell mixture that leucocyte will be captured gently be superimposed to from
In heart medium, by density gradient centrifugation, as a result can be divided into 4 layers, bottom is immune magnetic particle and leukocyte complexes, according to
Secondary is up centrifugation medium layer, tumor cells, buffer layer.Tumor cells are drawn according to purpose, may be remained immune
Magnetic particle, removes residual immunity magnetic particle by way of by magnetic frame, and the cell centrifugation for obtaining takes out bottom after bottom of the tube
Cell carries out smear.
4th, cell technique for fixing：Cell added cell fixer A after smear, in 25 DEG C~30 DEG C dryings 12~36 hours.
5th, hybridization in situ technique：After the cell after smear is completely dried, cell compartment is carried out using hybridization fixer B
Fixed, then aging, dewater treatment carries out in situ hybridization.Used probe is the CEP8 of biotin labeling, and hybridization instrument setting becomes
Degree warm in nature is 76 DEG C, and the time is 5min；Hybridization temperature is 37 DEG C, and the time is 1.5~3h.Hybridization is finished, and is taken out slide and is washed
Wash.
6th, Immunofluorescence technology：After the slide washing of in situ hybridization will have been completed, red quantum dot mark is prepared
The Streptavidin CD45 of note quantum dot-labeled with green is added to cell specimen dyeing liquor as immunofluorescence dyeing liquid
Area is washed in 37 DEG C of 1~3h of incubation after the completion of incubation, adds DAPI mountants to carry out mounting.
7th, tumour cell counting technology：The slide that in situ hybridization and Immunofluorescence test will have been completed is placed in fluorescence microscope
Under, burst of ultraviolel observation blueness DAPI signals, it may be determined that karyomorphism；Observation green CEP8 probe signals, it may be determined that cell
Hybridization signal point number in core；Observation red CD45 signals, determine leukocyte cell film staining conditions；It is final to determine that nucleus is
Monokaryon, probe signals point three and more than, the cell without CD45 staining signals is abnormal cell (tumour cell) and to count, whole
Slide is scanned, counts tumour cell number, tumour cell number as contained by the sample.
The present embodiment has found 103 for clinical samples are tested and analyzed, and has 27 healthy volunteers in 103 samples
(Healthy Controls), 34 pharynx nasalis benign lesion patients (Non-maniglant Disease) and 42 nasopharyngeal carcinoma
Patient, the circulating tumor cell detection data distribution of above clinical samples is provided by the present invention as shown in Fig. 2 as shown in Figure 2
The testing result of nasopharyngeal carcinoma circulating tumor cell detection kit is consistent with actual state.
Claims (5)
1. a kind of nasopharyngeal carcinoma circulating tumor cell detection kit, it is characterised in that by human peripheral leucocytes removal part and exempt from
Epidemic disease FISH identification part constitutes, and wherein human peripheral leucocytes removal part includes that dilution buffer, red blood cell are split
Solution liquid, density gradient centrifugation medium, cell fixer A and immunomagnetic beads, wherein erythrocyte cracked liquid is by ammonium chloride, bicarbonate
Sodium and EDETATE SODIUM solution are mixed, the antibody of the immunomagnetic beads anti-human LCA that has been covalent coupling
Magnetic bead, the antibody is CD45, and immunomagnetic beads particle diameter used is 0.2~1.5 μm；Cell fixer A functional components are alcohols；
Described immunofluorescence in situ hybridization identification part includes buffer solution, hybridizes fixer B, and the CEP8 of biotin labeling is visited
Pin, the CD45 antibody of red quantum dot mark, the quantum dot-labeled Streptavidin of green, mountant and bovine serum albumin(BSA)
Powder；The hybridization fixer B functional components are paraformaldehyde, and the CEP8 probes can be by the method for FISH
Probe mark is carried out to tumour cell, remaining leucocyte is dyeed the CD45 antibody of the red quantum dot mark and it is sent out
A length of 585nm~the 625nm of ejected wave, the quantum dot-labeled Streptavidin of green can be combined so as to DNA with CEP8 probes
Fragment is marked, and its launch wavelength is 525nm~545nm, and the mountant contains the fluorescence that can be combined with DNA strengths
Dyestuff DAPI.
2. nasopharyngeal carcinoma circulating tumor cell detection kit according to claim 1, it is characterised in that：Described red blood cell
The pH value of lysate is 7.0~8.0, and wherein the concentration of ammonium chloride is 0.5~2M, and sodium acid carbonate concentration is 10~200mM, EDTA
The concentration of disodium is 1~10mM.
3. nasopharyngeal carcinoma circulating tumor cell detection kit according to claim 1, it is characterised in that：Described biotin
The CEP8 probes of mark are purchased from branch and subsidiaries of Abbott Laboratories of the U.S..
4. nasopharyngeal carcinoma circulating tumor cell detection kit according to claim 1, it is characterised in that：Red quantum dot mark
The launch wavelength of the CD45 antibody of note is 605nm, and the launch wavelength of the quantum dot-labeled Streptavidin of green is 525nm, two
With albumen be attached quantum dot by the way of covalent coupling by person.
5. nasopharyngeal carcinoma circulating tumor cell detection kit according to claim 1, it is characterised in that：Described density level bands
The density for spending centrifugation medium is 1.02~1.5g/cm3, its active ingredient is sucrose, and sucrose mass concentration for 5%~
60%.
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| CN109439732A (en) * | 2018-12-25 | 2019-03-08 | 迈迪速能医学技术（天津）有限公司 | A kind of kit early sieved for three-dimensional noninvasive tumour |
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| CN111351937A (en) * | 2018-12-20 | 2020-06-30 | 上海细胞治疗集团有限公司 | MMR protein expression deletion detection kit and detection method thereof |
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| CN107907399A (en) * | 2017-11-24 | 2018-04-13 | 遵义医学院 | A kind of preparation method for the mountant that can mark nucleus at the same time |
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| CN111351937A (en) * | 2018-12-20 | 2020-06-30 | 上海细胞治疗集团有限公司 | MMR protein expression deletion detection kit and detection method thereof |
| CN109439732A (en) * | 2018-12-25 | 2019-03-08 | 迈迪速能医学技术（天津）有限公司 | A kind of kit early sieved for three-dimensional noninvasive tumour |
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| CN111024624A (en) * | 2019-12-20 | 2020-04-17 | 东南大学 | PARP-1 single particle detection method based on dark field scattering imaging |
| CN111471678A (en) * | 2020-04-29 | 2020-07-31 | 河南赛诺特生物技术有限公司 | Human chromosome 8 centromere probe and preparation method and kit thereof |
| CN114636820A (en) * | 2022-05-07 | 2022-06-17 | 珠海圣美生物诊断技术有限公司 | Kit and method for detecting circulating PD-L1 positive cells |
| CN114636820B (en) * | 2022-05-07 | 2022-10-21 | 珠海圣美生物诊断技术有限公司 | Kit and method for detecting circulating PD-L1 positive cells |
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