CN106834511A - A kind of kit of the breast cancer detection based on liquid biopsy - Google Patents
A kind of kit of the breast cancer detection based on liquid biopsy Download PDFInfo
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Abstract
The invention provides a kind of kit of the breast cancer detection based on liquid biopsy, the kit includes：Dyeing for strengthening Color strengthens liquid and the specific antibody with fluorescent staining mark；Wherein described specific antibody includes：ER Alpha antibodies, VIM antibody, CD45 antibody and HER2 antibody；The dyeing enhancing liquid includes surfactant that concentration is 0.001~1mg/mL.Kit of the invention, can effectively be enriched with target cell, and be confirmed whether the early stage patient from breast cancer, and carry out tumor classification.Meanwhile, being detected by three knurl marks increases nodule detection sensitivity and further by the accuracy for detecting guarantee detection of CEP8.In addition the present invention strengthens Color by dyeing enhancing liquid, various antibody with fluorescent staining mark is combined with target cell, and target cell is dyeed, and more preferably, fluorescence is stronger, and sharpness of border for Color.
Description
Technical field
The present invention relates to liquid biopsy field, the reagent of specifically a kind of breast cancer detection based on liquid biopsy
Box.
Background technology
Breast cancer is a kind of disease of serious threat women's health, and its incidence of disease accounts for the 23% of all tumours, in women
First place is occupied, fatal rate also occupies second in all cancers.According to statistics, in China, just have 30 per every 100,000 population of annual
~40 are diagnosed as patient with breast cancer, and it is 45~55 years old to be diagnosed as the average age of breast cancer, than west women more
It is young.Thus, just it is particularly important for the detection in the early screening and disease progression of breast cancer.
At present, for the early screening of breast cancer, the method based on liquid biopsy detection circulating tumor cell (CTC) has
Numerous unexistent advantages of traditional detection method, including noninvasive, quick, high accuracy for examination.CTC is a kind of spontaneous or because examining
Treat the tumour cell that operation is discharged into Peripheral Circulation by solid tumor primary tumor or MET.Because transfer is that cancer mutually shuts
The main cause died, and CTC is considered as the seed of transfer, thus to the detection of CTC new Tumor biomarkers discovery,
Tumor prognosis judge and individualized treatment aspect has very big application potential, are one of focuses of domestic and international tumor research.Arrive
So far, most of breast cancer CTC related research focuses primarily upon metastatic breast cancer (MBC) patient, and some are studied
Show similarly there is CTC in early-stage breast cancer (EBC) patient, its positive rate is 9.4~48.6%.And CTC in blood
Positive rate is also related to breast cancer early stage recurrence and low life cycle.In fact, research shows tumour in 1mm, in blood
Detection CTC, by detecting the presence of CTC in Peripheral Circulation, can be in the early detection cancer of cancer.Therefore, effective CTC
Capture, enrichment analysis method are critically important for the morning sieve based on CTC.
What although the CTC capture enrichment analysis platforms up to the present, based on EpCAM were still detected as CTC in MBC
Goldstandard, but the method if appropriate for the detection for EBC still suffer from dispute, and with EpCAM as biomarker for
There is false negative very high in the detection that the CTC of EMT occurs, the epithelium biomarker of this wide spectrum does not have organizing specific yet
Property, it is impossible to the specific tissue-derived of CTC is reviewed, and this point is significant for follow-up oncotherapy.Thus, it is found that
And be particularly important using the detection and analysis that the specific biomarker of some breast cancer tissues carries out CTC.
The day for announcing is on January 15th, 2014, and notification number passes through to disclose one kind in the Chinese patent of CN102313813B
The method that immunofluorescence dyeing is detected to rare cell, the method carries out three colors to leucocyte, rare cell and nucleus
Dyeing, then by fluoroscopic examination.But the method is only capable of by a kind of monoclonal antibody come the specific recognition rare cell, is easily caused
Missing inspection.Simultaneously as the heterogeneity of cell, the amount of antigen of each cell expression differs, and causes fluorescence intensity sometimes very faint,
It is difficult to differentiate under fluorescence microscope.
The content of the invention
The deficiency that the main object of the present invention is directed to prior art presence provides a kind of breast cancer based on liquid biopsy
The kit of detection, the quantity of CTC in peripheral blood can be detected using the kit, and Source Tracing, tumour point are carried out to the CTC
Type and early screening, while fluorescence display cell membrane sharpness of border, testing result sensitivity are high.
The present invention is achieved through the following technical solutions：
A kind of kit of the breast cancer detection based on liquid biopsy, the kit includes：For strengthening Color
Dyeing enhancing liquid and with fluorescent staining mark specific antibody；
Wherein described specific antibody includes：ER Alpha antibodies, VIM antibody, CD45 antibody and HER2 antibody；
The dyeing enhancing liquid includes surfactant that concentration is 0.001~1mg/mL.
The solvent of the dyeing enhancing liquid is biological buffer.
Preferably, the surfactant can be Triton X-100, dimethyl sulfoxide (DMSO), NP-40, dodecyl sulphate
Any one in sodium (SDS).It is furthermore preferred that the surfactant is Triton X-100 or SDS.Most preferably, the dye
Color enhancing liquid includes the SDS that concentration is 0.01~1mg/mL, and the solvent of dyeing enhancing liquid is conventional biological buffer, such as PBS
Buffer solution etc..
Preferably, lymphocyte separation medium and the immunomagnetic beads for removing leucocyte are also included in the kit.Drench
Bar cell separation liquid and immunomagnetic beads can be used in removing the interference of red blood cell and leucocyte, be easy to preferably be enriched with CTC.
It is furthermore preferred that the immunomagnetic beads for removing leucocyte is surface coupling antibody CD45, CD14 and CD15
Immunomagnetic beads in one or more, most preferably including whole above-mentioned immunomagnetic beadses.
Preferably, the specific antibody ER Alpha antibodies, VIM antibody and HER2 antibody are with least two different transmitted waves
Fluorescent staining mark long, and the fluorescent staining mark that carries of CD45 antibody and above-mentioned all fluorescent stainings marks not phase
Together.According to well known to a person skilled in the art in order to distinguish different specific antibodies, the present invention should use different fluorescence
Dye marker.But because the quantity of fluorescence microscope passage is limited, in generally making ER Alpha antibodies, VIM antibody and HER2 antibody
Two of which antibody is marked with same fluorescent staining.The present invention is preferably ER Alpha antibodies and HER2 antibody with same glimmering
Light dye marker.Fluorescent staining labeling dye can be using any commonly employed fluorescent dye in this area, as long as being excited by regulation
Optical wavelength can be distinguished.Because the launch wavelength of fluorescent staining mark is each different, therefore by fluorescence microscope in difference
Optical filter under can distinguish completely.Wherein, present invention preferably employs fluorescent staining labeled as Alexa Fluor series point
Son, cyanine dye.More preferably Alexa594, CY5 and Alexa488, wherein Alexa594 launch wavelengths are 618nm, CY5 hairs
The a length of 670nm of ejected wave, Alexa488 launch wavelengths are 519nm.CD45 antibody with fluorescent staining mark is preferably CD45-
Alexa594 (red), the ER Alpha antibodies with fluorescent staining mark are ER α-Alexa488 (green), are marked with fluorescent staining
HER2 antibody is HER2-Alexa488 (green), (naked eyes are invisible, show for VIM-CY5 for the VIM antibody with fluorescent staining mark
Micro mirror scanning is assigned to purple).
Preferably, the fluorescence probe for FISH can be the spy for chromosome fluorescence in-situ hybridization
Pin, such as CEP8 fluorescence probes.
Preferably, also include accounting for the poly- second two of dyeing enhancing liquid weight/mass percentage composition 0.1~3% in the dyeing enhancing liquid
Alcohol.Polyethylene glycol coordinates the Color that can further strengthen dyeing liquor with surfactant.
As well known to a person skilled in the art, by the specific antibody marked with fluorescent staining and cell be incubated from
And the process for carrying out fluorescent staining can be liquid dyeing or solid dyeing.To first need to carry out carefully if carrying out liquid and dyeing
The cell of born of the same parents' dyeing is resuspended into cell suspension, dyeing pretreatment, cell dyeing is then carried out successively, then cell is transferred into load glass
Carried out on piece cell fix, slide mounting.And if carrying out solid dyeing on slide, then first by cell on slide
Carry out cell to fix, after then carrying out dyeing pretreatment, cell dyeing successively, then to slide mounting.
Above-mentioned cell is fixed and carried out by cell fixer, and the cell fixer can be commonly used in the art thin
The combination of one or more in born of the same parents' fixer, such as paraformaldehyde, glutaraldehyde, formalin, ethanol, acetone.
The method for carrying out CTC detections using the kit of the above-mentioned breast cancer detection based on liquid biopsy includes following step
Suddenly：
Target cell in enrichment peripheral blood；
Enhancing dyeing pretreatment is carried out to target cell using dyeing enhancing liquid；
Fluorescent staining is carried out to target cell using the specific antibody marked with fluorescent staining；
Target cell is fixed；
FISH is carried out with fluorescence probe；
DAPI mountings and microscopy.
By the target cell after enhancing dyeing pretreatment, the combination of specific antibody and target cell is more preferable, fluorescence
Dyeing becomes apparent from, cell membrane sharpness of border.
As well known to a person skilled in the art, when tumor cell specific antigen is present in intracellular, should be to thin
After after birth padding and fixed target cell, then by cell membrane rupture of membranes and using the specific antibody pair with fluorescent staining mark
Target cell carries out intracellular Fluorescence dyeing.
Preferably, the target cell in the enrichment peripheral blood is comprised the following steps：
(1) by peripheral blood centrifugal separation plasma and haemocyte, and blood plasma is removed；
(2) to Cell Buffer and lymphocyte separation medium centrifugation layering is added in step (1), red blood cell layer is removed；
(3) to the immunomagnetic beads that is added in step (2) for removing leucocyte and incubation obtains suspension；
(4) the suspension Magneto separate in step (3) is removed into leucocyte and remaining red blood cell, then obtains richness after washing
Target cell after collection.
Described lymphocyte separation medium can be lymphocyte separation medium commonly used in the art, for according to Density Separation
Red blood cell, currently preferred use Ficoll separating liquids.
Enrichment of the present invention to target cell includes removing red blood cell twice, can be more clean to red blood cell removal, and
And by immunomagnetic beads, leucocyte and remaining red blood cell can be disposably removed, bioaccumulation efficiency is higher.Processed by above-mentioned enrichment
Interference of other cells for fluorescent staining can be excluded afterwards, be conducive to preferably being observed by fluorescence microscope.
Preferably, it is described using dyeing enhancing liquid target cell is carried out the process time of enhancing dyeing pretreatment for 5~
20 minutes.The final concentration of 0.2 μ g/mL~1mg/mL of surfactant after dyeing enhancing liquid addition.Wherein, dyeing enhancing liquid is excellent
The addition of choosing is 1~20 μ L.When the addition of dyeing enhancing liquid is more, the intensity for strengthening fluorescence is higher, but may
Cell membrane is caused to damage, stiffening effect is not again too obvious when addition is very few.
Preferably, it is described when carrying out fluorescent staining to target cell using the specific antibody marked with fluorescent staining, first
The specific antibody that will be marked with fluorescent staining is according to 1:The volume ratio of (100~200) is diluted with buffer solution, then by after dilution
Specific antibody mixes incubation with target cell.
Preferably, final concentration of 2~10 μ of specific antibody after the specific antibody with fluorescent staining mark is added
g/mL.Wherein, the specific antibody with fluorescent staining mark is according to 1:200 volume ratio is diluted with buffer solution.What is added is special
Property AC it is higher, more easily cause unspecific staining, influence the accuracy of result.
Above-mentioned buffer solution can be general biological cell buffer solution, such as PBS.It is furthermore preferred that PBS
It is the phosphate buffer of 0.01M, wherein Na2HPO4、NaCl、KH2PO4With the concentration of KCl be respectively 8mM, 136mM, 2mM and
2.6mM, pH value is 7.2-7.4.
Inventive principle
In primary breast cancer, about 75~80% show as the estrogen receptor alpha positive (ER α+), the state pair of ER α
All play an important roll in the classification of breast cancer and direction of medication usage.The Determines of ER α patient with breast cancer is if appropriate for hormone
Treatment, and this treatment method is still at present ER α+main auxiliary treating method of patient with breast cancer.So based on ER α's
CTC detections not only have the ability of tissue specificity positioning, and its expression status also has necessarily for the treatment of breast cancer
Directive significance.Additionally, human epidermal growth factor receptor 2 (HER2) is also a kind of important breast cancer predicting marker, in original
About 10~30% show as HER2+ in hair property breast cancer, and related to poor prognosis and wellability higher.Additionally,
About 11% breast cancer shows as ER α -/HER2+, and so combining the detection sensitivity of ER α+and HER2+ can reach about 80%, and
And the primary breast cancer patients of part HER2-, it is likely to that the CTC of HER2+ can be produced during development.
In addition to the breast cancer of above-mentioned ER α+or HER2+, base type breast cancer often show as three negative (TNBC) (ER α -/
PR-/HER2-), such breast cancer CTC cannot then use above-mentioned marker detection.Although TNBC only accounts for 15% of breast cancer or so,
But because it has transfer ability and poor prognosis higher, so can not be ignored.In this regard, vimentin (VIM) wide expression
In the breast cancer tissue for not relying on hormone, be used for very early detect hormone receptor-negative base type breast cancer, its
Detection sensitivity in such breast cancer can reach about 94%, it is possible to used as a life of good TNBC breast cancer CTC
Thing mark.
No. 8 chromosome abnormalities are present in various solid tumors, including prostate cancer, oophoroma, kidney, breast cancer, knot are straight
Intestinal cancer etc., the situation using No. 8 chromosome abnormalities in the method detection cell or tissue of FISH (FISH) is wide
It is general in clinical diagnosis, the present invention to be aided with No. 8 X chromosome centrics domain while based on tumor markers biopsy
(CEP8) FISH detection checkings, further by the detection of CEP8 while the detection of double knurl marks increases nodule detection sensitivity
Ensure the specificity of detection.Wherein, CEP8 probes are the fluorescence probe for being marked with SpectrumOrange, all thin for selecting
No. 8 cells of numerical abnormalities of chromosomes are target cell in born of the same parents.By fluorescence microscope, regulation excitation light wave is a length of
SpectrumOrange launching lights are 588nm orange lights during 559nm, can be entered with above-mentioned tumor markers and leucocyte mark fluorescence
Row detects that the detection counted out by specific orange light probe determines No. 8 numbers of chromosome of target cell simultaneously.The present invention exists
While based on tumor markers biopsy, the FISH for the being aided with CEP8 detection checkings that can be selected increase CTC in the detection of three knurl marks
Detection while detection sensitivity further by CEP8 ensures the specificity of detection.
Accordingly, tri- biological marker analyte detection breast cancer CTC of joint ER α, HER2 and VIM have sensitivity very high, this
Outward can also accordingly to breast cancer CTC partings：ER α+it is tube chamber type breast cancer (Luminal)；HER2+/ER α-it is the HER2 positives
Breast cancer (HER2+)；ER α -/HER2-/VIM+ is base type breast cancer (Basal).The CTC for detecting can be according to ER α and HER2
Expression whether determine whether from mammary gland, and combine the expression of VIM and can carry out parting to breast cancer, while tracing to the source
Realize the early screening to breast cancer.
The present invention uses the CD45 antibody with Alexa594 fluorescent stainings mark to leukocyte surface specific stain, with band
The ER Alpha antibodies and HER2 antibody of Alexa488 fluorescent stainings mark, and the VIM antibody with CY5 fluorescent stainings mark is to capture
Circulating tumor cell carry out specific stain.By fluorescence microscope, Alexa594 when regulation excitation wavelength is 591nm
Launching light is the partially orange feux rouges of 618nm；CY5 launching lights are that (naked eyes are invisible, show for 670nm far-red lights when excitation wavelength is 650nm
Micro mirror scanning is assigned to purple)；Alexa488 launching lights are 519nm green glows when excitation wavelength is 499nm.So different by adjusting
Excitation wavelength and the time for exposure, it can be observed that the different fluorescence marked on different cells, and then target cell can be entered
Row is distinguished：CD45+ is leucocyte；HER2+ or ER α+it is tumour CTC cells, wherein HER2+/ER α+can determine that it derives from breast
The patient of gland cancer, and further parting can also be carried out to CTC according to the expression of different specific antigens：ER α+or HER2
+ it is tube chamber type breast cancer (Luminal) or HER2 positive breast cancers (HER2+)；And ER α -/HER2-/VIM+ is base type mammary gland
Cancer (Basal).
The various antibody with fluorescent staining mark of selection are conducive to confirming the particular type of target cell, but when antibody kind
When class is more than one, the combination of target cell and antibody is easy for being disturbed, and causes fluorescent staining not good, influences fluorescence microscope
Observation.And the Membrane protein antigen determinant that dyeing enhancing liquid of the invention passes through exposed cell surface, mark band fluorescent staining
Antibody be easier to combine on the target protein of cell surface, so as to strengthen the fluorescence intensity of mark cell membrane, and make cell
Membrane boundary is clear.
The kit of the breast cancer detection based on liquid biopsy of the invention, can effectively be enriched with target cell, and
Confirm whether the target cell of enrichment derives from the early stage patient of breast cancer, and carry out tumor classification.Meanwhile, the present invention passes through
The detection of three knurl marks increases nodule detection sensitivity and further by the accuracy for detecting guarantee detection of CEP8.In addition this hair
It is bright by dye enhancing liquid strengthen Color, make it is various with fluorescent staining mark antibody can be with target cell target protein
With reference to, target cell is dyeed, more preferably, fluorescence is stronger, and sharpness of border for Color.So can simultaneously strengthen haematogenous
Cell surface specificity fluorescent dyes the fluorescent staining effect with tumor cell specific mark so that CTC is thin with haematogenous
Born of the same parents are easier to distinguish, and reduce the false positive of detection, improve the specificity of detection.Also, the inventive method is simple, low cost, tool
There is very strong practicality.
Brief description of the drawings
Fig. 1 is the fluorescent staining detection Merge figures of MCF7 MCF-7s；
Fig. 2 is the fluorescent staining detection Merge figures of SK-BR-3 MCF-7s；
Fig. 3 is the fluorescent staining detection Merge figures of MDA-MB-231 MCF-7s；
Fig. 4 is the fluorescent staining detection Merge figures after tumour cell capture.
Specific embodiment
By the following specific examples further illustrate the invention：The experiment of unreceipted actual conditions in the following example
Method, conventionally and condition, or selects according to catalogue.
Ficoll-Paque PLUS of the lymphocyte separation medium from GE healthcare companies.
CD45 immunomagnetic beadses are from Thermo Fisher Scientific'sCD45。
Alexa 594, Alexa 488 and CY5 fluorescent dyes come from Thermo Fisher Scientific.
CEP 8SpectrumOrange DNA Probe Kit of the CEP8 probes from Abbott.
Target cell in the enrichment peripheral blood of embodiment 1
(1) peripheral blood is centrifuged to remove plasma protein：By 8.5mL peripheral bloods in horizontal centrifuge 800g, normal temperature from
Heart 7min, supernatant discarded.
(2) to the PBS and the lymphocyte separation medium of 3mL that 5~6mL is added in the blood plasma of step (1), in centrifugation
450g in machine, normal temperature centrifugation 7min.It is divided into three layers after centrifugation, red bottom is red blood cell layer, the intermediate layer master of whitish
To be leucocyte and CTC etc., the upper strata of yellow is blood plasma, draws whole liquid more than red blood cell layer, remove the red thin of bottom
Born of the same parents' layer.
(3) there is the immunomagnetic beads of CD45 antibody to being added dropwise over the coupling of 200mL surfaces in step (2), on horizontal shaker
Incubation obtains suspension, and horizontal shaker rotating speed is arranged on 120-150rpm, inclines 20-30 °, normal temperature, level shake 20min.
(4) suspension in step (3) is adsorbed into 2min with big magnetic frame.Careful absorption is not attached to the liquid on tube wall
Body, remove leucocyte and remaining red blood cell, with PBS wash and it is resuspended after be enriched with after target cell.
Target cell after 2 pairs of enrichments of embodiment carries out fluorescent staining
(1) enhancing dyeing pretreatment：Enhancing liquid, normal temperature are dyeed to 2 μ L are added in the target cell after the enrichment of about 50 μ L
Stand 10min.The dyeing enhancing liquid is the PBS solution of SDS or Triton X-100, and SDS concentration is 0.1mg/mL.
(2) cell surface dyeing：After the μ L of CD45-Alexa 5941 are diluted with the PBS of 199 μ L, step is added to
Suddenly in the cell suspension after the completion of (1) pretreatment, then lucifuge is incubated 20min.Add PBS cleaning cellular liquid after incubation,
950g is centrifuged 4min, removes supernatant to 100 μ L.
(3) cell is fixed：Cell transfer in step (2) is applied on slide, fixer poly first is subsequently adding
Aldehyde, fixed 10min, PBS washings slide 2 times, each 5min.
(4) cell rupture of membranes：Cell compartment is added dropwise the μ L of cell rupture of membranes liquid 200 on slide after cell fixation, is incubated 10min
Afterwards plus PBS cleaning slide 2 times, each 5min.The cell rupture of membranes liquid is molten for the PBS of Triton X-100
Liquid, Triton X-100 concentration is 0.5%.
(5) cell intracellular mark dyeing：Each 1 μ L of ER α-Alexa488, Her2-Alexa 488 and VIM-CY5 are used
After the PBS dilution of 198 μ L, cell compartment on above-mentioned slide is added to, then lucifuge is incubated 20min, then adds PBS bufferings
Liquid cleaning slide 2 times, each 5min.
(6) FISH：To the CEP8 fluorescence probes that 10 μ L are added dropwise on slide, covered, surrounding is sealed up
Piece glue mounting.The prehybridization 10min at 76 DEG C, then hybridizes 4h at 37 DEG C.
(7) DAPI mountings and microscopy：Tear mounting glue off, cleaned with PBS 2 times (5min/ times), and slough lid glass
Piece, 10 μ L mountants mountings is added after natural drying and (wherein the content of mountant is DAPI to covered：Glycerine=1:
9), finally with Nikon DS-U3 fluorescence microscopes, microscopy condition is：Alexa594 transmittings during excitation light wave a length of 591nm
Light is 618nm feux rouges, time for exposure 100ms；CY5 launching lights are that (naked eyes are or not 670nm far-red lights during excitation light wave a length of 650nm
It can be seen that, micro- scarnning mirror is assigned to purple), time for exposure 100ms；Alexa488 launching lights are during excitation light wave a length of 499nm
519nm green glows, time for exposure 100ms；DAPI launching lights are 455nm blue lights during excitation light wave a length of 345nm, the time for exposure 10~
20ms；SpectrumOrange launching lights are 588nm orange lights during excitation light wave a length of 559nm, and as a result time for exposure 100ms shows
Show, the most presentation CD45 positive stainings of the leucocyte that normal human peripheral blood is separate, knurl mark negative staining, FISH results are
CEP8 dliploids, do not have knurl mark positive or CEP8 numerical abnormalities cell.
The fluorescent staining detection of the cell line of embodiment 3
MCF7 (ER α +/HER2-) or SKBr3 (ER α -/HER2+) or MDA-MB-231 (ER α -/HER2-/VIM+) is thin
Born of the same parents system (source Chinese Academy of Sciences cell bank is bought), takes 10 after enzymolysis, digestion5Individual cell, about 50 μ L, according to the step of embodiment 2
Suddenly cell fluorescence dyeing and fluorescence microscopy are carried out.Microscopy condition is：Alexa594 transmittings during excitation light wave a length of 591nm
Light is 618nm feux rouges, time for exposure 100ms；CY5 launching lights are that (naked eyes are or not 670nm far-red lights during excitation light wave a length of 650nm
It can be seen that, micro- scarnning mirror is assigned to purple), time for exposure 100ms；Alexa488 launching lights are during excitation light wave a length of 499nm
519nm green glows, time for exposure 100ms；DAPI launching lights are 455nm blue lights during excitation light wave a length of 345nm, the time for exposure 10~
20ms, as a result as Figure 1-3.
From Fig. 1-3 as can be seen that the cell of each cell line shows the nucleus of blue light, explanation under 358nm exciting lights
There is cell；Red is not shown under 591nm exciting lights, illustrates that CD45 is negative, in the absence of leucocyte.
Under 499nm exciting lights, MCF7 cells and the display of SKBr3 cells are green, illustrate that ER α or HER2 are positive；MDA-
MB-231 cells do not show green, illustrate that ER α and HER2 are negative.
Under 650nm exciting lights, MDA-MB-231 cells display purple illustrates that VIM is positive, MCF7 cells and SKBr3
Cell does not show purple, illustrates that VIM is negative.
The tumour cell capture rate of embodiment 4 is detected
Collection healthy volunteer's 8.5ml blood samples, take MCF7, SK-BR-3 and MDA-MB-231 MCF-7 difference
It is 31,26 and 26 addition blood samples.Then according to embodiment 1 the step of carries out tumor cell enrichment, according still further to the step of embodiment 2
Rapid fluorescent staining and the fluorescence microscopy for carrying out tumour cell.Microscopy condition is：During a length of 591nm of excitation light wave
Alexa594 launching lights are 618nm feux rouges, time for exposure 100ms；CY5 launching lights are that 670nm is remote during excitation light wave a length of 650nm
Feux rouges (naked eyes are invisible, and micro- scarnning mirror is assigned to purple), time for exposure 100ms；Alexa488 during excitation light wave a length of 499nm
Launching light is 519nm green glows, time for exposure 100ms；DAPI launching lights are 455nm blue lights, exposure during excitation light wave a length of 345nm
10~20ms of time；SpectrumOrange launching lights are 588nm orange lights, time for exposure during excitation light wave a length of 559nm
100ms, as a result as shown in Figure 4.
Result shows that tumour cell (ER α+or Her2+ or VIM+) captures number 69, capture rate 83.13%.ER α or
The Her2 positive (ER α+or Her2+) cell numbers 49, positive rate 59.04%；The VIM positive (VIM+) cell numbers 20, sun
Property rate 24.1%；Knurl mark is positive (ER α+or Her2+/VIM+) tumor cell number 0, positive rate 0%；Non- two times of CEP8
Body (CEP8 ≠ 2) tumor cell number 37, non-dliploid rate 44.58%.
It can be seen from the results above that using kit of the invention carry out based on liquid biopsy CTC detection relative to
For existing single knurl mark detection, the tumour cell sensitivity in capture blood is higher.Simultaneously according to the Different Results of detection, can
To carry out the parting of breast cancer.And according to the detection of CEP8, the specificity of detection can be increased, meet different detection mesh
's.
Embodiment described above is a kind of preferably scheme of the invention, not makees any formal to the present invention
Limitation, for those skilled in the art, is not departing from embodiment of the present invention principle and claim
Under the premise of, some improvements and modifications can also be made, these improvements and modifications are also considered as the protection domain of the embodiment of the present invention.
Claims (8)
1. a kind of kit of the breast cancer detection based on liquid biopsy, it is characterised in that：The kit includes：For strengthening
The dyeing enhancing liquid of Color and the specific antibody with fluorescent staining mark；
Wherein described specific antibody includes：ER Alpha antibodies, VIM antibody, CD45 antibody and HER2 antibody；
The dyeing enhancing liquid includes surfactant that concentration is 0.001~1mg/mL.
2. the kit of the breast cancer detection based on liquid biopsy according to claim 1, it is characterised in that：The dyeing
The solvent for strengthening liquid is biological buffer.
3. the kit of the breast cancer detection based on liquid biopsy according to claim 1, it is characterised in that：The surface
Activating agent is selected from any one in TritonX-100, dimethyl sulfoxide (DMSO), NP-40, lauryl sodium sulfate.
4. the kit of the breast cancer detection based on liquid biopsy according to claim 1, it is characterised in that：The reagent
Also include lymphocyte separation medium and the immunomagnetic beads for removing leucocyte in box.
5. the kit of the breast cancer detection based on liquid biopsy according to claim 4, it is characterised in that：It is described to be used for
The immunomagnetic beads of leucocyte is removed for surface is coupled one or more had in the immunomagnetic beads of antibody CD45, CD14 and CD15.
6. the kit of the breast cancer detection based on liquid biopsy according to claim 1, it is characterised in that：It is described special
Property the fluorescent staining of antibody ER Alpha antibodies, VIM antibody and HER2 antibody with least two different emissions mark, and
The fluorescent staining mark that CD45 antibody is carried is differed with above-mentioned all fluorescent staining marks.
7. the kit of the breast cancer detection based on liquid biopsy according to claim 1, it is characterised in that：The reagent
Also include the fluorescence probe for FISH in box.
8. the kit of the breast cancer detection based on liquid biopsy according to claim 1, it is characterised in that：The dyeing
Also include accounting for the polyethylene glycol of dyeing enhancing liquid weight/mass percentage composition 0.1~3% in enhancing liquid.
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| CN201710168601.7A CN106834511B (en) | 2017-03-21 | 2017-03-21 | Breast cancer detection kit based on liquid biopsy |
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| CN112979805A (en) * | 2019-12-13 | 2021-06-18 | 桐庐纳泰医学检验实验室有限公司 | VEGFR2 fluorescence labeled antibody for circulating tumor cell detection, and detection method and application thereof |
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