CN105823878A - Kit for detecting phenotypes of circulating tumor cells - Google Patents
Kit for detecting phenotypes of circulating tumor cells Download PDFInfo
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Abstract
The invention provides a kit for detecting phenotypes of circulating tumor cells. The kit comprises an antibody-oligonucleotide probe obtained by respectively coupling E-cadherin, Cadherin-11 and EpCAM antibodies with different oligonucleotides, an amplification primer and buffer solution used for performing PCR amplification on the antibody-oligonucleotide probe. The kit also comprises a fluorescence probe. According to the kit disclosed by the invention, the E-cadherin, Cadherin-11 and EpCAM are taken as markers to perform parallel detection on content of each antigen in CTC, so that the content of each phenotype in CTC is analyzed, and each phenotype of CTC is distinguished.
Description
Technical field
The present invention relates to immunization detection field, specifically one be used for detecting circulating tumor
The test kit of cell phenotype.
Background technology
Circulating tumor cell (CTC) refer to spontaneous or because of operation of diagnosis and treatment by solid tumor primary tumor or
Metastasis discharges into the tumor cell of Peripheral Circulation, typically with individual cells or cell mass
Presented in (also known as circulating tumor microemboli, CTM) in blood circulation.Transfer is
The main cause of cancer related mortality, and CTC is considered the seed of transfer.CTC is in order to obtain
Obtaining mobility and aggressive, the phenotype that can lose some epithelial cell (includes that form, surface resist
Former, gene expression etc.) and obtain the phenotype of some mesenchymal cell, here it is epithelial-mesenchymal
Change (EMT, Epithelial-MesenchymalTransition).Most of malignant tumor cell
EMT is there is during departing from primary tumor.Therefore there is different phenotype in CTC, including on
Epidermis type, interstitial phenotype and intermediate phenotype.Phenotype analytical is for finding tumor, judging transfer feelings
Condition, judging prognosis, treatment situation etc. are the most significant.Study discovery recently, CTM with
Interstitial phenotype CTC is closely related, and a high proportion of interstitial phenotype CTC has with chemotherapy resistance
Close (Science 2013；339:580).Research additionally shows, CTM can resist mistake nest
Apoptosis (anoikis), resistant cells cytotoxic drug (Journal of Clinical
Oncology,2012；30:525), than single tumor cell, there is higher metastatic potential
(Clinical Cancer Research,2001；7:4080).Therefore, CTM and Interstitial cell table
Type CTC (EMT CTC), may be more with the dependency of prognosis compared with single common CTC
By force.
The method of existing differentiation CTC phenotype mainly have by density, molecular size or based on
Biological marker EpCAM (EpCAM) etc..By density, molecular size
It is easily caused CTC leakage sieve, and only can detect that epithelial phenotype CTC based on EpCAM, with
Time normal cell express identical mark EpCAM and can cause false positive, and phenotype heterogeneity meeting
False negative, result is caused to be inaccurate.
Meanwhile, CTC concentration in peripheral blood is the lowest, and therefore detection CTC needs is quick
Sensitivity is higher.The use in conjunction of many marks antibody can be effectively improved detection sensitivity and specificity
(Journal of the National Cancer Institute,2009,101(1):61-66).In order to examine
Survey the phenotype of CTC, it would be desirable to be able to distinguish marker and the detection platform of CTC phenotype.
Summary of the invention
The main object of the present invention is that the not enough offer existed for prior art is a kind of for examining
Survey CTC phenotype test kit, this test kit can efficiently and accurately detect epithelial phenotype,
Interstitial phenotype and the CTC of intermediate phenotype, detection sensitivity is high simultaneously, operates rapid and convenient.
The present invention is achieved through the following technical solutions: one is used for detecting circulating tumor cell
The test kit of phenotype, it includes:
By E-cadherin, Cadherin-11 and EpCAM antibody respectively from different oligonucleoside
The antibody-oligonucleotide acid probe that acid coupling obtains；
For above-mentioned antibody-oligonucleotide acid probe being carried out amplimer and the buffering of PCR amplification
Liquid；
Also include fluorescent probe；
Further, described antibody-oligonucleotide acid probe includes oligonucleotide probe part and resists
Body portion；Described oligonucleotide probe part be the 5' end of oligonucleotide is carried out aldehyde group modified after
Obtaining, described antibody moiety obtains after antibody is carried out diazanyl modification；Described oligonucleoside
Acid probe part and described antibody moiety obtain described antibody-oligonucleotide acid probe through coupling.
Further, described oligonucleotide probe part is held 5 ' with amido modified
Oligonucleotide, with the SFB that molar equivalent is 5-20 times carry out aldehyde group modified after obtain；Institute
State antibody moiety be the SANH that antibody molar equivalent is 10-50 times is carried out diazanyl modification after
Obtain.
Wherein, above-mentioned SFB refers to 4-carbamoyl benzoate N-succinimide ester；Above-mentioned
SANH refers to 4-(N-maleimidomethyl) hexamethylene-1-carboxylic acid succinimide ester.
Described antibody-oligonucleotide acid probe is to be (7-10) by mol ratio: the oligonucleotide probe of 1
Part and antibody moiety, reaction obtained after 4-24 hour at ambient temperature.
Preferably, a length of 16-22nt of described oligonucleotide, 5 ' ends are 6-14nt length
Primer recognition sequence, 3 ' end extension primers carry out extending extension.
Further, a length of 50-80nt of described extension primer, its 3 ' end ends are with few
3 ' end complementary pairings of nucleotide, 5 ' ends can form hairpin structure, and intermediate sequence and fluorescence
The complementary of probe.The sequence extending primer is preferably shown in SEQ ID NO:14.
Preferably, the sequence of described oligonucleotide is appointing shown in SEQ ID NO:1-13
One.
Described amplimer includes a pair for antibody-oligonucleotide acid probe is carried out PCR expansion
The forward of increasing, reverse primer, 5 ' ends of described forward primer are complementary with oligonucleotide.
Preferably, described circulating tumor cell is that cervical cancer, breast carcinoma or carcinoma of prostate circulation are swollen
One or more in oncocyte.
Inventive principle
E-cadherin antigen is expressed in most of epithelial tissue.The expression of E-cadherin is adjusted
Control plays an important role during EMT.When CTC from epithelial phenotype to interstitial Phenotype Transition time,
The expression of E-cadherin reduces.And optionally E-cadherin can cause human carcinomas group
That knits dedifferentes and aggressiveness.E-cadherin is at epithelial phenotype CTC and intermediate phenotype CTC
In be exist, but be non-existent in interstitial phenotype CTC.This be one well
Marker, is used for distinguishing epithelial phenotype CTC/ intermediate phenotype CTC and interstitial phenotype CTC.
EpCAM antigen is the glycoprotein of a surface of cell membrane, high in epithelial cancer cell
Degree is expressed, and low expression in normal epithelium cell.EpCAM is usually in interstitial phenotype CTC
Middle down-regulated expression, but in epithelial phenotype CTC and intermediate phenotype CTC, all there is expression.
Cadherin-11 antigen is a kind of cell adhesion molecule, usually in osteoblast and tumor
Cell is expressed.Cadherin-11 is had in intermediate phenotype CTC and interstitial phenotype CTC.
Therefore Cadherin-11 can be as distinguishing epithelial phenotype CTC and intermediate phenotype CTC/ interstitial
The foundation of phenotype CTC.In the CTC of carcinoma of prostate, the expression of Cadherin-11 is higher.
The expression of antigen such as document " Nature Reviews Clinical in various phenotypes CTC
Oncology》,2014Jul；Disclosed in 11 (7): 401-12, by by E-cadherin,
Cadherin-11 and EpCAM, as marker, can carry out the analysis of each phenotype in CTC.
There is advantages that
Test kit the most of the present invention is by by E-cadherin, Cadherin-11 and EpCAM
As marker, carry out the content of each antigen in Parallel testing CTC, thus analyze and obtain CTC
In the content of each phenotype, distinguish each phenotype of CTC.
2. the present invention is by the way of coupling aldehyde radical and diazanyl obtain antibody-oligonucleotide acid, can make
Standby obtain above-mentioned antibody respectively with three kinds of probes of oligonucleotide coupling, every kind of antibody-oligonucleotide
The oligonucleotide of acid probe is different from.Such that it is able to expanded by the PCR of oligonucleotide,
Carry out the antigenic content that in Parallel testing CTC, each antibody is the most corresponding.
3. with extending the primer 3 ' the end amplifications to oligonucleotide, it is ensured that oligonucleotide chain is too short
Time PCR expand requirement, and extend the hairpin structure that primer 5 ' holds and can increase the heap of base
Block power, thus strengthen probe acute, make universal antibody-oligonucleotide acid probe detect more
Sensitive.
4. hold complementary pairing with extending primer 3 ' due to oligonucleotide, therefore extend extension
Process can react voluntarily in PCR amplification procedure, anti-with after extending again after extending extension
Body-oligonucleotide is that template expands, single step reaction, and reaction efficiency is high.
5. owing to have employed the mode of PCR amplification, than the sensitivity of conventional ELISA method detection
Degree improves several order of magnitude, even if the concentration of CTC is the lowest, it is also possible to enter trace antigen
Row detection.
Detailed description of the invention
By the following specific examples further illustrate the invention: unreceipted in the following example
The experimental technique of actual conditions, conventionally and condition, or selects according to catalogue.
Present invention antibody-oligonucleotide to be obtained acid probe, its be by first by oligonucleotide with anti-
Body carries out the directed modification of aldehyde radical and diazanyl respectively and obtains oligonucleotide part and antibody moiety, then
Carry out hydrazone key coupling to obtain.Preferably, described oligonucleotide probe part is with ammonia by 5 ' ends
Base modify oligonucleotide, with the SFB that molar equivalent is 5-20 times carry out aldehyde group modified after must
Arriving, preferably molar equivalent is 10 times；Described antibody moiety is
The SANH of 10-50 times obtains after carrying out diazanyl modification, and preferably molar equivalent is 25 times.Its
In, SFB is 4-carbamoyl benzoate N-succinimide ester, and SANH is 4-(N-maleimide
Ylmethyl) hexamethylene-1-carboxylic acid succinimide ester.With on 5 ' ends of SFB modified oligonucleotide
Amino can obtain aldehyde radical activation oligonucleotide, hydrazine can be obtained with SANH modified antibodies
The antibody protein of base activation.The two reaction is available for public technology, such as China's document
" immuno-chip antibody based on making nucleic acid molecular hybridization fixes new method ", " analytical chemistry ", 2013
Year the 2nd phase, Sha Sha etc. is disclosed.The condition such as response time, reaction temperature can be according in document
Hold to make and well known to a person skilled in the art adjustment.Currently preferred technical scheme is: by 5 '
Hold and dissolve with the amido modified PBS that oligonucleotide pH value is 7.4, then
Mix with the SFB solution that molar equivalent is oligonucleotide 10 times, room temperature reaction 2.5 hours,
Repurity obtains aldehyde group modified oligonucleotide probe part.It is 7.4 by antibody pH value
After PBS dilution, mix with the SANH solution that molar equivalent is antibody 25 times, room temperature
Reacting 2.5 hours, repurity obtains the antibody moiety that diazanyl is modified.By mol ratio it is finally
(7-10): the oligonucleotide probe part of 1 and antibody moiety at room temperature coupling reaction obtains institute
State antibody-oligonucleotide acid probe.
1 be further described by the following examples, remaining unaccounted actual conditions and
Experimental technique, conventionally and condition, or selects according to catalogue.
" room temperature " described in the present embodiment refers to the indoor temperature of routine, generally 15-30
℃。
Sequence is SEQ ID NO:(1-13 by the present embodiment) Oligo be abbreviated as Oligo (1-13),
Oligo is oligonucleotide.
PBS in the present embodiment is phosphate buffer, and MES buffer is 2-(N-
Quinoline generation) ethanesulfonic acid buffer.
QPCR in the present embodiment is real-time fluorescence quantitative PCR.
Nanodrop in the present embodiment is spectrophotometer.
BCA method in the present embodiment refers to the quantitative approach of determination of protein concentration.
Buffer used by the present embodiment is: the PBS of differently configured pH value, at this
In the detailed description of the invention of invention, specifically include the PBS that pH value is 6.0, and
PH value is the PBS of 7.4, and configures the MES buffer that pH value is 5.0,
Filtration sterilization processes, and deposits for 4 DEG C.
Embodiment 1E-cadherin-oligonucleotide probe
(1) oligonucleotide probe is aldehyde group modified
By the Oligo1 (oligonucleotide) of the 50nmol phosphoric acid buffer of the pH 7.4 of 0.1M
Liquid is configured to solution.Weigh the SFB (4-carbamoyl benzoate N-succinimide ester) of 500nmol,
After dissolving by dry DMF (DMF), at room temperature reaction 2.5h, cross post
Purification obtains Oligo-FB (aldehyde group modified oligonucleotide).
The concentration of detection Oligo-FB: detect A with Nanodrop spectrophotometer260Value,
The concentration calculating Oligo-FB is 0.65nmol/ μ L.
Detect aldehyde group modified rate: repair with quantitative 2-hydrazine pyridine-2-HCI solution detection aldehyde radical
Decorations rate.Take above-mentioned Oligo-FB and join in 2-hydrazine pyridine-2-HCI solution, after vibration mixing
React 1h in 37 DEG C, be 1.31 with the light absorption value at Nanodrop detection 360nm, calculate
Its modification rate, A360Under modification rate be 0.87.
(2) diazanyl of antibody E-cadherin is modified
Antagonist E-cadherin is dense with Nanodrop detection antibody protein after carrying out desalting and purifying
Degree is 6.5mg/mL.
Diluting antibody E-cadherin with the phosphate buffer that pH value is 7.4 is 2 to concentration
mg/mL.Weigh the SANH (antibody is 1:25 with the mol ratio of SANH) of 25 times amount, molten
Solution, in dry DMF, is then added in antibody, at room temperature reaction 2.5h, crosses column purification and obtains
To E-cadherin-SANH (E-cadherin that diazanyl is modified).
The concentration of detection E-cadherin-SANH: calculated by BCA method after modifying
The concentration of E-cadherin-SANH is 1.60mg/mL.
Detection diazanyl modification rate: modify with quantitative 2-formyl benzene sulfonyl sodium salt solution detection diazanyl
Rate.Take in the 2-formyl benzene sulfonyl sodium salt solution that antibody-SANH after purification joins, vortex
After mixing, the light absorption value at 37 DEG C of reaction 1h, Nanodrop detection 348nm is 0.36.
Calculated by the densitometer of the light absorption value at 348nm and E-cadherin-SANH
The diazanyl modification rate of E-cadherin-SANH is 3.2.
(3) coupling of Oligo-FB Yu E-cadherin-SANH
Oligo-FB with E-cadherin-SANH is mixed according to mol ratio 7:1 mixing vortex, room
Temperature reaction 4h, the product finally obtained i.e. can get described universal after crossing column purification
E-cadherin-Oligo probe.
Take E-cadherin-Oligo probe and carry out Nanodrop detection.Have bright under 354nm
Aobvious absworption peak occurs, the coupling success of Oligo-FB Yu E-cadherin-SANH is described.
Take E-cadherin-Oligo probe and carry out SDS-PAGE detection, analyze E-cadherin with
Oligo coupling degree, with pure E-cadherin compares, obtains a plurality of electrophoretic band, explanation
The E-cadherin upper coupling oligonucleotide of varying number.
Take E-cadherin-Oligo probe and carry out BCA method Concentration Testing.BCA method Concentration Testing
Calculate the concentration of conjugate antibody.With in the quantitative E-cadherin-Oligo of strand quantification kit
The concentration of Oligo1.The ratio of E-cadherin Yu Oligo1 can be calculated, it is possible to and repair
Decorations rate results contrast.Illustrate Oligo1 and the coupling of antibody in E-cadherin-Oligo molecule
Ratio, result is as shown in table 1.
Oligo and the coupling ratio of antibody in table 1 E-cadherin-Oligo molecule
The QPCR specific amplification detection of embodiment 2Oligo molecule
It is 25000 molecular number/μ l, 83333 molecular number by Oligo1-13 molecular dilution to concentration
/ μ l and 250000 molecular number/μ l tri-concentration.Then as a example by Oligo1-3, by Oligo1-3
Mix under same concentrations, obtain biased sample A, B, C, as shown in table 2.
Table 2 embodiment 2 sample formulations
Above-mentioned sample is configured QPCR according to table 3 and expands liquid, obtain QPCR amplification kit,
Then according to the program shown in table 4 carries out QPCR amplification to Oligo1-3, and to aggregate sample
Oligo1-3 in product A, B, C each carries out QPCR amplification, after respective extension extension
Oligo1-3 is template, and the Ct value of amplification is as shown in table 5.Wherein, extending primer is
RT-P, its 3 ' end ends and 3 ' end complementary pairings of oligonucleotide, 5 ' ends can be formed
Hairpin structure, and the complementary of intermediate sequence and MGB probe (MGty), this reality
Execute the RT-P of example by sequence for as a example by shown in SEQ ID NO:14.Forward primer is FP, instead
It is RP to sequence.FP is by sequence for as a example by shown in SEQ ID NO:15, and RP is with sequence as SEQ
As a example by shown in ID NO:16, MGty by sequence for as a example by shown in SEQ ID NO:17,5 ' ends
Fluorophor FAM labelling, 3 ' ends are for MGB.
The QPCR amplification kit of table 3 Oligo molecule
| Reagent | 1 part of consumption (μ l) | Final concentration |
| Nuclear free water | 1.35 | |
| RT-P(1uM) | 0.25 | 25nM |
| FP(10uM) | 0.3 | 300nM |
| RP(10uM) | 0.3 | 300nM |
| MGty(10uM) | 0.1 | 100nM |
| Premix Ex Taq(2×) | 5 | |
| Oligo | 2.5 |
Table 4 QPCR amplification program
Table 5 amplification Ct value
| Oligo1 | Oligo2 | Oligo3 | |
| 25000 molecular number/μ l | 25.33 | 25.92 | 23.98 |
| 83333 molecular number/μ l | 23.50 | 24.17 | 22.43 |
| 250000 molecular number/μ l | 21.96 | 22.54 | 20.60 |
| A | 25.25 | 25.88 | 24.03 |
| B | 23.40 | 24.08 | 22.27 |
| C | 21.81 | 22.56 | 20.69 |
As can be seen from Table 5, the Oligo1-3 in biased sample A, B, C, in same concentrations
The Ct value of lower amplification is about 0.1 with the amplification Ct value of single Oligo.Meanwhile, according to every
Regression straight line drawn by the variable concentrations sample of Oligo, calculates to obtain regression beeline equation and correlation coefficient
R2 is as follows:
Oligo1:y=-3.3664x+40.11, R2=0.99937；
Oligo2:y=-3.384x+40.809, R2=0.99997；
Oligo3:y=-3.3824x+38.928, R2=0.99463.
The A-C sample that every corresponding for Oligo is calculated according to separate equation, calculates score
Subnumber is divided by theory of correspondences molecular number, and gained detection efficiency is as follows:
Therefore, expanding Ct difference between parallel sample is about 0.1, and detection efficiency is
Between 98.5%-110.5%, illustrate that three Oligo do not have non-specific expansion with remaining two respectively
Increase, do not interfere with amplification efficiency each other.Remaining Oligo is identified can be tied equally
Really, illustrate between a plurality of Oligo of this experimental design without cross influence, it is ensured that multiplex PCR
Specificity, accuracy and sensitivity.Other Oligo can obtain same conclusion, due to
Length reason describes in detail the most one by one.
Embodiment 3 makes standard curve
Same result can be obtained with Oligo1-13, in order to simplify process, following example
As a example by Oligo1-3.
According to the step of embodiment 1, with the SFB that molar equivalent is 5 times, Oligo2 is repaiied
Decorations obtain Oligo-FB, with the SANH that molar equivalent is 10 times, EpCAM are carried out diazanyl and repair
Decorations obtain EpCAM-SANH, are Oligo-FB and EpCAM-SANH of 10:1 by mol ratio
Reaction obtained EpCAM-Oligo2 after 16 hours at ambient temperature.
According to the step of embodiment 1, with the SFB that molar equivalent is 20 times, Oligo3 is carried out
Modification obtains Oligo-FB, carries out Cadherin-11 with the SANH that molar equivalent is 50 times
Diazanyl is modified and is obtained Cadherin-11-SANH, by Oligo-FB that mol ratio is 8:1 with
Cadherin-11-SANH obtains after reacting 24 hours at ambient temperature
Cadherin-11-Oligo3。
By E-cadherin-Oligo1, EpCAM-Oligo2 and Cadherin-11-Oligo3 according to table
The test kit of 3 and the program of table 4 carry out QPCR amplification, to extend the Oligo1-3 after extending
For template, the amplification obtained individually expands consistent with Oligo1-3, and the antibody of coupling is described
Part expands not impact to the PCR of Oligo.
With standard dilutions by E-cadherin-Oligo1, EpCAM-Oligo2 and
Cadherin-11-Oligo3 is each diluted to concentration: 833,2500,8333,25000,
83333 and 250000 molecular number/2.5 μ l, totally six concentration, blank group is deionized water.
According to the recipe configuration QPCR amplification kit shown in table 3, then according to shown in table 4
Program to E-cadherin-Oligo1, EpCAM-Oligo2 of above each concentration and
Cadherin-11-Oligo3 carries out QPCR amplification, with the Oligo1-3 after extension extension as template,
Amplification is as shown in table 6:
Table 6 standard curve QPCR amplification Ct value
| Concentration (molecular number/2.5 μ l) | E-cadherin-Oligo1 | EpCAM-Oligo2 | Cadherin-11-Oligo3 |
| 0 | 33.59 | 32.43 | 33.41 |
| 833 | 32.72 | 30.38 | 31.6 |
| 2500 | 31.48 | 29.15 | 30.17 |
| 8333 | 29.74 | 27.56 | 29.26 |
| 25000 | 28.19 | 25.86 | 27.09 |
| 83333 | 26.32 | 23.95 | 24.68 |
| 250000 | 24.64 | 22.22 | 21.82 |
The Ct value of the variable concentrations according to antibody-Oligo, draws standard curve, linearly returns
Return calculating, obtain regression beeline equation and correlation coefficient is as follows:
E-cadherin-Oligo1:y=-3.2951x+42.554, R2=0.9978；
EpCAM-Oligo2:y=-3.3332x+40.384, R2=0.9961；
Cadherin-11-Oligo3:y=-3.872x+43.542, R2=0.9659.
The QPCR detection of embodiment 4CTC cellular antigens
Take the peripheral blood blood of various cancers patient, the CTC amount of antigen in blood is examined
Survey.Require that experimenter's routine blood test wbc value is positioned at 2 × 106～1.2 × 107Between individual/mL,
And blood sample is in processing procedure, there is not following abnormal phenomena in sample: as sample is red
After cell cracking not exclusively causes sample cytoadherence, sample erythrocyte splitting, remaining cell is on the low side
/ leukocyte is on the low side, and haemolysis or clot occurs in whole blood sample.And experimenter's relevant information
Completely, sample collection, store method specification, experimental implementation specification, specifically comprise the following steps that
(1) take various cancers patient blood 3ml, add 12mL cell pyrolysis liquid, run gently
Fully mix.Then it is centrifuged after sample is placed in 2-8 DEG C of refrigerator cracking 15min and abandons supernatant,
Add 10mLPBS washed cell；It is centrifuged and abandons supernatant, add 400 μ LPBS re-suspended cells.
(2) after adding 100 μ l blocking buffer, room temperature closes 20min；Add
After E-cadherin-Oligo1, EpCAM-Oligo2 and Cadherin-11-Oligo3 probe, in room
Temperature terminates after hatching 40min reacting and being centrifuged removing supernatant.
(3) 120ul eluent mix homogeneously, ice are added after using PBS washed cell 3 times
On hatch 2min, centrifuging and taking 100ul supernatant, the unreacted probe of eluting；It is eventually adding 20
μ L reaction neutralizer neutralizes.
The QPCR condition detection that standard curve is identical is made by embodiment 3
The Ct value of E-cadherin-Oligo1, EpCAM-Oligo2 and Cadherin-11-Oligo3 probe,
Result is as shown in table 7:
The QPCR testing result of table 7 CTC
The Immunofluorescence test of embodiment 5 CTC antigen
In order to detect the reliability of test kit testing result of the present invention, to the same as in Example 4
Blood carries out Immunofluorescence test, and its step is as follows:
(1) take patient blood 3ml the same as in Example 4, add 12mL cell pyrolysis liquid,
Reverse fully mixing gently.Then it is centrifuged after sample is placed in 2-8 DEG C of refrigerator cracking 15min
Abandon supernatant, add 10mLPBS washed cell；It is centrifuged and abandons supernatant, add 400 μ LPBS weights
Outstanding cell.
(2) use CD45 magnetic bead to remove part leukocyte, after being centrifuged, use 100ul PBS weight
Outstanding cell.
(3) the fixing rear smear of paraformaldehyde solution that concentration is 4% is added, after PBS washes three times
Closing 30 minutes, PBS washes three times again.
(4) anti-E-cadherin, EpCAM and Cadherin-11 for each labelling it is separately added into
Antibody, PBS washes three times in 4 DEG C of wet boxes overnight.
(5), after DAPI contaminates core mounting, counting, result such as table 8 are identified with fluorescence microscope
Shown in:
The Immunofluorescence test result of table 8 CTC
From the testing result of table 7 and table 8 it can be seen that the lowest sample of Ct value, contain is anti-
Original molecule number is the highest.And microscopy gained fluorescent positive CTC number is the most, i.e. E-cadherin,
EpCAM and Cadherin-11 content is the most, show Ct value and both microscopy numbers in negative correlation,
Concordance is good.
The Cadherin-11 antigen presentation amount of patient A is low, illustrates that CTC is in epitheliated type.B、
The expression of E-cadherin, EpCAM and Cadherin-11 antigen of patient D is the most slightly higher,
Illustrate that CTC is in middle type.And the expression of the E-cadherin antigen of C, E patient is relatively
Low, and the EpCAM antigen presentation amount of patient E is the lowest, illustrates that they are in CTC's
The characteristic of epithelial cell is gradually lost, and converts for interstitial type from epitheliated type through EMT.
The patient numbering above-mentioned A-E carries out postoperative tracking, the result of postoperative recurrence such as table 9 institute
Show:
Table 9 postoperative recurrence result
| Patient number | Postoperative recurrence |
| A | Without recurrence Clinical signs |
| B | Without recurrence Clinical signs |
| C | 3 months after operation recurs |
| D | Without recurrence Clinical signs |
| E | 3 months after operation recurs |
By table 9 it can be seen that patient's 3 months after operation of C, E group recurs.With C, E group
It is consistent that interstitial type CTC of patient has higher metastatic potential with invasive ability result.
For those skilled in the art, former without departing from the embodiment of the present invention
On the premise of reason, it is also possible to make some improvements and modifications, these improvements and modifications are also considered as this
The protection domain of inventive embodiments.
Claims (9)
1. the test kit being used for detecting circulating tumor cell phenotype, it is characterised in that: its bag
Include:
By E-cadherin, Cadherin-11 and EpCAM antibody respectively from different oligonucleoside
The antibody-oligonucleotide acid probe that acid coupling obtains；
For above-mentioned antibody-oligonucleotide acid probe being carried out amplimer and the buffering of PCR amplification
Liquid；
Also include fluorescent probe.
Test kit for detecting circulating tumor cell phenotype the most according to claim 1,
It is characterized in that: described antibody-oligonucleotide acid probe includes oligonucleotide probe part and antibody
Part；Described oligonucleotide probe part be the 5' end of oligonucleotide is carried out aldehyde group modified after
Arriving, described antibody moiety obtains after antibody is carried out diazanyl modification；Described oligonucleotide
Probe portion and described antibody moiety obtain described antibody-oligonucleotide acid probe through coupling.
Test kit for detecting circulating tumor cell phenotype the most according to claim 2,
It is characterized in that: described oligonucleotide probe part is with amido modified oligonucleoside by 5 ' ends
Acid, with the SFB that molar equivalent is 5-20 times carry out aldehyde group modified after obtain；Described antibody
Part be the SANH that antibody molar equivalent is 10-50 times is carried out diazanyl modification after obtain.
Test kit for detecting circulating tumor cell phenotype the most according to claim 1,
It is characterized in that: described antibody-oligonucleotide acid probe is to be (7-10) by mol ratio: the oligonucleoside of 1
Acid probe part and antibody moiety, reaction obtained after 4-24 hour at ambient temperature.
Test kit for detecting circulating tumor cell phenotype the most according to claim 1,
It is characterized in that: a length of 16-22nt of described oligonucleotide, 5 ' ends are 6-14nt length
Primer recognition sequence, 3 ' end extension primers carry out extending extension.
Test kit for detecting circulating tumor cell phenotype the most according to claim 5,
It is characterized in that: a length of 50-80nt of described extension primer, its 3 ' end ends and few core
3 ' end complementary pairings of thuja acid, 5 ' ends can form hairpin structure, and intermediate sequence is visited with fluorescence
The complementary of pin.
Test kit for detecting circulating tumor cell phenotype the most according to claim 1,
It is characterized in that: the sequence of described oligonucleotide is arbitrary shown in SEQ ID NO:1-13
Individual.
Test kit for detecting circulating tumor cell phenotype the most according to claim 1,
It is characterized in that: described amplimer includes a pair for being entered by antibody-oligonucleotide acid probe
The forward of performing PCR amplification, reverse primer, 5 ' ends of described forward primer are mutual with oligonucleotide
Mend.
Test kit for detecting circulating tumor cell phenotype the most according to claim 1,
It is characterized in that: described circulating tumor cell is that cervical cancer, breast carcinoma or carcinoma of prostate circulation are swollen
One or more in oncocyte.
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