CN107475202A - A kind of lung cancer circulating tumor cell capture and Activity determination, analysis method and its application - Google Patents
A kind of lung cancer circulating tumor cell capture and Activity determination, analysis method and its application Download PDFInfo
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Abstract
The invention provides a kind of capture of lung cancer circulating tumor cell and Activity determination,Analysis method and its application,On the basis of negative sense enrichment lung cancer CTCs,Pass through the capture to lung cancer CTCs,Capture is on filter membrane,Lung cancer CTCs is identified by joint-detection karyomorphism and three kinds of specific mRNAs expression quantity,Nucleus size and morphological feature analysis are carried out to the doubtful CTCs captured from every blood sample,Differentiate whether be TTF1 positive lung cancers CTCs with reference to TTF1 mRNA and pan CK mRNA,And then the TTF1 positive lung cancers CTCs of proliferation activity or TTF1 positive lung cancers CTCs without proliferation activity is classified as according to whether MKI67 mRNA expression is positive,The detection method high sensitivity of the invention,High specificity,Efficient capture and lung cancer patient peripheral blood T TF1 positive CTCs quantity can be counted out,It is capable of detecting when TTF1 positives CTCs activity,And analyze each cell whether there is proliferation activity.
Description
Technical field
The present invention relates to lung cancer liquid biopsy field, and in particular to a kind of lung cancer circulating tumor cell capture is examined with activity
Survey, analysis method and its application.
Background technology
Circulating tumor cell (Circulating tumor cells, CTCs) detection is the master in tumour liquid biopsy field
One of content is wanted, but existing circulating tumor cell detection technique only relies on some feature that tumour cell has, in tumour
Capture CTCs in the peripheral blood of patient, carry out simple count, be the shortcomings that these detection techniques：On the one hand specificity is lacked,
The cell identified in peripheral blood may not be exactly tumour cell, at present not for the specific detection side of TTF1 positive lung cancers
Method；What is more important lacks the in-depth analysis to CTCs, it is impossible to obtains CTCs proliferation activity information, which can not be identified
CTCs has transfer invasive ability, and which CTCs has been enter into the apoptosis stage.
Existing lung cancer CTCs detection techniques have three kinds：
The first technology is to ratify to assume that for clinical Cell search technologies, its general principle by U.S. FDA
Tumor cell surface expresses epithelial cell adhesion molecule (EpCAM), using the immunomagnetic beads of coupling EpCAM antibody from whole blood
Forward direction capture CTCs, the expression of intracellular epitheliated type mark CK albumen is then captured using immunofluorescence technique detection,
If the cell CK protein positives of capture, can be determined as CTC by the cell.
Second of technology is to be used for the peripheral blood folate receptor-positive cell of clinical lung cancer patient by Chinese cFDA certifications
Detection technique, its general principle are the foundation a number of folacin receptors of lung carcinoma cell surface expression, will be red in whole blood sample
After cell and leucocyte removal, there are the oligonucleotides probe specific bond lung cancer CTCs surfaces of folate ligand using coupling
Folacin receptor, the quantity of probe is then quantitatively detected using Real-Time round pcrs, so as to calculate folacin receptor indirectly
Quantity, finally by the total quantity of folacin receptor estimate corresponding to CTCs numbers.
The third technology is that after negative sense is enriched with CTCs, whole cells of acquisition are entered under the background that a small amount of leucocyte be present
Row special marking, the cell with certain feature is CTCs, and can be divided into not according to the feature difference that CTCs has
Same type.The expression quantity using the intracellular epitheliated types of probe in detecting CTC and interstitial type mark is specifically included, if the main tables of CTC
Epitheliated type CTC is then judged as up to epitheliated type mark；Interstitial type CTC is judged as if mainly expression interstitial type mark；
The two has concurrently, is judged as mixed type CTC.
Existing all kinds of lung cancer circulating tumor cell (CTCs) detection techniques respectively have shortcoming above.To use EpCAM antibody
For core CTCs forward direction beneficiation technologies the shortcomings that be that sensitivity is low, it is impossible to the CTCs of EpCAM molecules is not expressed in capture；To remove
The shortcomings that leucocyte is the CTCs negative sense beneficiation technologies of core is poor specificity, and the epithelium non-tumour source in blood is thin
Born of the same parents, vascular endothelial cell etc. are mistaken for CTC.In addition, existing lung cancer CTCs detection techniques are not all to CTCs propagation
Activity is measured, it is impossible to which the CTCs for illustrating to detect is to be in apoptosis stage or multiplicative stage.On the whole, it is existing
Low sensitivity, specific deficiency be present and the shortcomings that lack this three aspect of activity identification in CTCs detection techniques.
Therefore, invent a kind of detection method high sensitivity, high specificity, efficient capture and can count out outside lung cancer patient
TTF1 positives CTCs quantity in all blood, is capable of detecting when TTF1 positives CTCs activity, and analyzes each cell whether there is propagation
The capture of lung cancer circulating tumor cell and Activity determination, analysis method and its application of activity, have extensive market prospects.
The content of the invention
In view of the shortcomings of the prior art, the present invention, which provides, invents a kind of detection method high sensitivity, high specificity, Neng Gougao
Effect captures and counts out lung cancer patient peripheral blood T TF1 positive CTCs quantity, is capable of detecting when TTF1 positives CTCs activity,
And analyze each cell whether there is proliferation activity lung cancer circulating tumor cell capture with Activity determination, analysis method and its should
With.
The technical proposal of the invention is realized in this way：
A kind of lung cancer circulating tumor cell capture and activity test method, it is characterised in that：
Comprise the following steps：
(1) lung cancer CTCs capture：
1) peripheral blood is gathered：Routinely venous blood is adopted in operation, collects 5ml peripheric venous bloods；
2) crack and remove red blood cell：5ml peripheric venous bloods in step 1) are added in 45ml erythrocyte cracked liquids,
The uninterrupted jog 20min of room temperature after reverse mixing, with abundant splitting erythrocyte；500g centrifuges 10min, removes supernatant；Use cell
Cell precipitation is resuspended in cleaning solution, and 1000g centrifuges 10min again, removes supernatant；
3) leucocyte is removed：Using magnetic bead combination leucocyte, cell precipitation is resuspended using 500 μ l cell washing solutions, will
In CD45 antibody coating magnetic bead addition step 2) in cell suspension, it is put on rotary mixer and fully combines, 150r/min room temperatures
20min is reacted, after magnetic frame removes leucocyte, obtains the lung cancer CTCs cell suspensions for remaining a small amount of leucocyte；
4) miillpore filter retention lung cancer CTCs：The lung cancer CTCs cell suspensions obtained in step 3) are added into 50 μ L, 40%
After neutral formalin solution fixes 8min, transfer cell suspension is in filter of the aperture for 8 μm of miillpore filters, by negative
The less leukoreduction filter of diameter in cell suspension is fallen in pressure suction, and the aperture that lung cancer CTCs is more than filter membrane because of diameter is trapped within
On filter membrane, lung cancer CTCs cells are obtained, it is standby；
(2) TTF1 positive lung cancers CTCs identification：
1) cell is fixed：1ml, 4% neutral formalin are added in the lung cancer CTCs cells that miillpore filter captures in step (1)
Solution fills the fully fixed 1h of room temperature, and negative pressure leaching removes fixer, and after adding 1ml cell washing solutions washing 2min, suction filtration eliminates
Removing residue formaldehyde, obtain fixed lung cancer CTCs cells；
2) permeabilization：The filter membrane containing fixed lung cancer CTCs cells in above-mentioned steps is transferred in 24 orifice bores, added
0.1%Triton X-100 permeabilization agent, 5min being reacted at room temperature, increase membrane passage, cell washing solution washes film 3 times,
Permeabilization agent is thoroughly removed, obtains the lung cancer CTCs cells after permeabilization；
3) digest：Lung cancer CTCs cells after permeabilization are soaked in 2.5 μ g/ml pepsin working solutions, are kept for 40 DEG C
30min is incubated, film is washed 3 times using cell washing solution, thoroughly removes pepsin, mRNA probes is entered intracellular, obtains
The lung cancer CTCs cells of digestion；
4) rna probe hybridization binding purpose mRNA molecules：Use three species specificity mRNA probes and intracellular target
MRNA molecules fully hybridize combination, 40 DEG C of 2~3h of incubation, using the washing of probe cleaning solution three times, remove uncombined RNA and visit
Pin；
5) side chain DNA technique expands：On the basis of specific probe and target mRNA specific bonds, using branch chain DNA skill
Art amplifies hybridization signal, applies three kinds of different amplified fluorescence systems to three kinds of mRNA probe complexes respectively, final to cause often
The kind peculiar fluorescence labeling of mRNA molecules is shown in cell in-situ；
6) nuclear targeting：Transfer is loaded with lung cancer CTCs miillpore filter and slide, adds DAPI fluorescent staining liquid marks
Remember nucleus, neutral gum mounting standby microscopy；
7) full-automatic fluorescence microscopy scarnning mirror and image is recorded：Each cell is scanned respectively using multichannel fluorescence microscope
4 kinds of fluorescence signals, 4 kinds of fluorescence signals are respectively from DAPI (mark nucleus), Cy3 (mark TTF1 mRNA), FITC (marks
Remember pan-CK mRNA) and Cy5 (marking MKI67 mRNA) fluorescent dye, to lung cancer CTCs cells according to certain after the end of scan
Standard carry out activity analysis.
A kind of activity assays of lung cancer circulating tumor cell, it is characterised in that：
Described TTF1 positive lung cancer CTCs Analysis of test results includes：To each cell captured from blood sample,
Karyomorphism feature, three kinds of mRNA expressions of results are analyzed successively；
First, standard is read to each cell to sort out：
(1) it is single by inspection three kinds of mRNA expression of results interpretation standards of cell：
Positive interpretation standard is expressed by inspection cell TTF1 mRNA：Fluorescence points >=3 under Cy3 passages；
Negative interpretation standard is expressed by inspection cell TTF1 mRNA：Fluorescence points ＜ 3 under Cy3 passages；
Positive interpretation standard is expressed by inspection cell pan-CK mRNA：Fluorescence points >=3 under FITC passages；
Negative interpretation standard is expressed by inspection cell pan-CK mRNA：Fluorescence points ＜ 3 under FITC passages；
Positive interpretation standard is expressed by inspection cell MKI67 mRNA：Fluorescence points >=3 under Cy5 passages；
Negative interpretation standard is expressed by inspection cell MKI67 mRNA：Fluorescence points ＜ 3 under Cy5 passages；
(2) TTF1 positive lung cancers CTCs interpretation standards：
For inspected each cell, if meeting three conditions in following criterion simultaneously, for TTF1 positive lungs
Cancer CTCs；
TTF1 positive lung cancer CTCs interpretation standards are：
1) cell nuclear diameter is more than neutrophil leucocyte and lymphocyte in leucocyte；
2) TTF1 mRNA are expressed as the positive；
3) pan-CK mRNA are expressed as the positive；
(3) TTF1 positive lung cancers CTCs proliferation activity interpretation standards：
There is the TTF1 positive lung cancers CTC of proliferation activity：Meeting the premise of TTF1 positive lung cancers tri- interpretation conditions of CTCs
Under, MKI67 mRNA are expressed as the positive；
TTF1 positive lung cancers CTC without proliferation activity：Meeting the premise of TTF1 positive lung cancers tri- interpretation conditions of CTCs
Under, MKI67 mRNA are expressed as feminine gender；
(4) testing result of each blood sample of analysis and summary：
According to above-mentioned interpretation standard, nucleus size and form are carried out to the doubtful CTCs captured from every blood sample
Signature analysis is learned, differentiates whether be TTF1 positive lung cancer CTCs with reference to TTF1 mRNA and pan-CK mRNA, and then according to MKI67
Whether mRNA expression is positive to be classified as the TTF1 positive lung cancers CTCs of proliferation activity or the TTF1 positives without proliferation activity
Lung cancer CTCs.After each cell analysis finishes, the TTF1 positive lung cancers CTCs for collecting lung source in the blood sample is total
Number, and collect the TTF1 positive lung cancer CTCs numbers, percentage and the nothing in lung source that have proliferation activity in lung source respectively
The TTF1 positive lung cancer CTCs numbers of proliferation activity, percentage.
Described mRNA probe combinations are：Pan-CK mRNA, TTF1 mRNA and MKI67mRNA.
Described erythrocyte cracked liquid is：0.8%NH4Cl, 0.1%KHCO3With 0.1mM EDTA.
The cell washing solution is：8.0g/L NaCl、0.2g/L KCl、1.44g/L Na2HPO4And 0.24g/L
KH2PO4。
The probe cleaning solution is：0.15M NaCl, 0.015M sodium citrates and 25% formamide.
A kind of capture of lung cancer circulating tumor cell and detection, analysis method, use mRNA probe combinations and cell karyomorphism
State carries out activity classification to lung cancer CTCs.
A kind of capture of lung cancer circulating tumor cell and Activity determination, analysis method, CTCs points applied to patients with lung cancer
Class.
A kind of capture of lung cancer circulating tumor cell and Activity determination, analysis method, applied to the noninvasive auxiliary of patients with lung cancer
Help diagnosis.
The present invention has following good effect：Existing all kinds of lung cancer circulating tumor cell (CTCs) detection sensitivities or
Shortcoming on specificity all be present, the use of the shortcomings that CTCs forward direction beneficiation technologies of the EpCAM antibody as core to be that sensitivity is low, no
The lung cancer CTCs for not expressing EpCAM molecules can be captured；The shortcomings that to remove CTCs negative sense beneficiation technologies of the leucocyte as core, exists
In poor specificity, the epithelial cell in non-tumour source, vascular endothelial cell etc. in blood are mistaken for lung cancer CTCs.
Detection method provided by the invention is higher than positive beneficiation technologies based on CTCs capture rates have been recognized at present
On negative sense beneficiation technologies, it ensure that the sensitivity of detection method is sufficiently high；The present invention is using nucleus is larger, pan-CK simultaneously
The cell that positive two features of mRNA expression can ensure to detect is non-blood borne cell, cell in lung cancer patient blood sample
The cell that TTF1 mRNA expression positive classifications can prompt to detect is lung tissue derived cell, and comprehensive nucleus is larger, pan-CK
MRNA expression is positive can be ensured in lung cancer patient blood sample with positive three features of TTF1 mRNA expression while have these three features
Cell be TTF1 positive lung cancer CTCs, the specificity that ensure that this detection method is used in combination in these three features；In a word, on
State three being used in combination for feature and ensure that the present invention high sensitivity, high specificity on detection TTF1 positive lung cancers CTCs.
Existing lung cancer CTCs detection methods, it can not all detect TTF1 positive lung cancers CTCs proliferation activity.The present invention makes
It can measure whether TTF1 positive lung cancers CTCs expresses MKI67 mRNA with MKI67 mRNA detection probes, according to MKI67
Whether TTF1 positive lung cancers CTCs effectively can be carried out proliferation activity classification by the mRNA expression positive.
For the present invention on efficiently concentrating lung cancer CTCs miillpore filter negative sense beneficiation technologies, joint-detection nucleus is big
Small, pan-CK mRNA, TTF1 mRNA and MKI67 mRNA, efficient capture and lung cancer patient peripheral blood T TF1 can be counted out
Positive CTCs quantity, and TTF1 positives CTCs activity is capable of detecting when, having in lung cancer patient blood sample can be bred and lived
Property TTF1 positive lung cancers CTCs quantity and the quantitative measure without proliferation activity TTF1 positive lung cancers CTCs come out, and are follow-up TTF1
The studies and clinical application of positive lung cancer provides a kind of new detection technique.
Therefore, in summary：The invention detection method high sensitivity, high specificity, efficient capture and lung can be counted out
TTF1 positives CTCs quantity in carninomatosis human peripheral, is capable of detecting when TTF1 positives CTCs activity, and analyzes each cell
Whether there is proliferation activity.
Brief description of the drawings
Fig. 1 is the present invention to single lung cancer CTCs captures and proliferation activity classification results example.
Patients with lung cancer CTCs is counted for the present invention by Fig. 2 and proliferation activity classification results example.
Embodiment
Embodiment
Prepare in advance first following：
Erythrocyte cracked liquid, is formulated and is：0.8%NH4Cl, 0.1%KHCO3With 0.1mM EDTA；
Cell washing solution formula is：8.0g/L NaCl、0.2g/L KCl、1.44g/L Na2HPO4And 0.24g/L
KH2PO4；
Probe washs formula of liquid：0.15M NaCl, 0.015M sodium citrates and 25% formamide；
MRNA combination probes include：Pan-CK mRNA bonding probes, TTF1 mRNA bonding probes and MKI67 mRNA are tied
Close probe；
A kind of lung cancer circulating tumor cell capture and activity test method,
Comprise the following steps：
(1) lung cancer CTCs capture：
1) peripheral blood is gathered：Routinely venous blood is adopted in operation, collects 5ml peripheric venous bloods；
2) crack and remove red blood cell：5ml peripheric venous bloods in step 1) are added in 45ml erythrocyte cracked liquids,
The uninterrupted jog 20min of room temperature after reverse mixing, with abundant splitting erythrocyte；500g centrifuges 10min, removes supernatant；Use cell
Cell precipitation is resuspended in cleaning solution, and 1000g centrifuges 10min again, removes supernatant；
3) leucocyte is removed：Using magnetic bead combination leucocyte, cell precipitation is resuspended using 500 μ l cell washing solutions, will
In CD45 antibody coating magnetic bead addition step 2) in cell suspension, it is put on rotary mixer and fully combines, 150r/min room temperatures
20min is reacted, after magnetic frame removes leucocyte, obtains the lung cancer CTCs cell suspensions for remaining a small amount of leucocyte；
4) miillpore filter retention lung cancer CTCs：The lung cancer CTCs cell suspensions obtained in step 3) are added into 50 μ L, 40%
After neutral formalin solution fixes 8min, transfer cell suspension is in filter of the aperture for 8 μm of miillpore filters, by negative
The less leukoreduction filter of diameter in cell suspension is fallen in pressure suction, and the aperture that lung cancer CTCs is more than filter membrane because of diameter is trapped within
On filter membrane, lung cancer CTCs cells are obtained, it is standby；
(2) TTF1 positive lung cancers CTCs identification：
1) cell is fixed：1ml, 4% neutral first are added in the lung cancer CTCs cells that miillpore filter captures in step (1)
Aldehyde solution fills the fully fixed 1h of room temperature, and negative pressure leaching removes fixer, and after adding 1ml cell washing solutions washing 2min, suction filtration removes
Removing residue formaldehyde to the greatest extent, the lung cancer CTCs cells after must fixing；
2) permeabilization：The filter membrane containing fixed lung cancer CTCs cells in above-mentioned steps is transferred in 24 orifice bores, added
0.1%Triton X-100 permeabilization agent, 5min being reacted at room temperature, increase membrane passage, cell washing solution washes film 3 times,
Permeabilization agent is thoroughly removed, obtains the lung cancer CTCs cells after permeabilization；
3) digest：Permeabilization lung cancer CTCs cells in above-mentioned steps are soaked in 2.5 μ g/ml pepsin working solutions,
40 DEG C of incubation 30min are kept, film is washed 3 times using cell washing solution, thoroughly removes pepsin, mRNA probes is entered thin
Intracellular, obtain postdigestive lung cancer CTCs cells；
4) rna probe hybridization binding purpose mRNA molecules：Use three species specificity mRNA probes and intracellular target
MRNA molecules fully hybridize combination, 40 DEG C of 2~3h of incubation, using the washing of probe cleaning solution three times, remove uncombined RNA and visit
Pin；
5) side chain DNA technique expands：On the basis of specific probe and target mRNA specific bonds, using branch chain DNA skill
Art amplifies hybridization signal, applies three kinds of different amplified fluorescence systems to three kinds of mRNA probe complexes respectively, final to cause often
The kind peculiar fluorescence labeling of mRNA molecules is shown in cell in-situ；
6) nuclear targeting：Transfer is loaded with lung cancer CTCs miillpore filter and slide, adds DAPI fluorescent staining liquid marks
Remember nucleus, neutral gum mounting standby microscopy；
7) full-automatic fluorescence microscopy scarnning mirror and image is recorded：Each cell is scanned respectively using multichannel fluorescence microscope
4 kinds of fluorescence signals, 4 kinds of fluorescence signals are respectively from DAPI (mark nucleus), Cy3 (mark TTF1 mRNA), FITC (marks
Remember pan-CK mRNA) and Cy5 (marking MKI67 mRNA) fluorescent dye, to lung cancer CTCs cells according to certain after the end of scan
Standard carry out activity analysis.
The target of 4 kinds of fluorescence signal detections
A kind of activity assays of lung cancer circulating tumor cell：
Described TTF1 positive lung cancer CTCs Analysis of test results includes：To each cell captured from blood sample,
Karyomorphism feature, three kinds of mRNA expressions of results are analyzed successively；
First, standard is read to each cell to sort out：
(1) it is single by inspection three kinds of mRNA expression of results interpretation standards of cell：
Positive interpretation standard is expressed by inspection cell TTF1 mRNA：Fluorescence points >=3 under Cy3 passages；
Negative interpretation standard is expressed by inspection cell TTF1 mRNA：Fluorescence points ＜ 3 under Cy3 passages；
Positive interpretation standard is expressed by inspection cell pan-CK mRNA：Fluorescence points >=3 under FITC passages；
Negative interpretation standard is expressed by inspection cell pan-CK mRNA：Fluorescence points ＜ 3 under FITC passages；
Positive interpretation standard is expressed by inspection cell MKI67 mRNA：Fluorescence points >=3 under Cy5 passages；
Negative interpretation standard is expressed by inspection cell MKI67 mRNA：Fluorescence points ＜ 3 under Cy5 passages；
(2) TTF1 positive lung cancers CTCs interpretation standards：
For inspected each cell, if meeting three conditions in following criterion simultaneously, for TTF1 positive lungs
Cancer CTCs；
TTF1 positive lung cancer CTCs interpretation standards are：
1) cell nuclear diameter is more than neutrophil leucocyte and lymphocyte in leucocyte；
2) TTF1 mRNA are expressed as the positive；
3) pan-CK mRNA are expressed as the positive；
(3) TTF1 positive lung cancers CTCs proliferation activity interpretation standards：
There is the TTF1 positive lung cancers CTC of proliferation activity：Meeting the premise of TTF1 positive lung cancers tri- interpretation conditions of CTCs
Under, MKI67 mRNA are expressed as the positive；
TTF1 positive lung cancers CTC without proliferation activity：Meeting the premise of TTF1 positive lung cancers tri- interpretation conditions of CTCs
Under, MKI67 mRNA are expressed as feminine gender；
(4) testing result of each blood sample of analysis and summary：
According to above-mentioned interpretation standard, nucleus size and form are carried out to the doubtful CTCs captured from every blood sample
Signature analysis is learned, differentiates whether be TTF1 positive lung cancer CTCs with reference to TTF1 mRNA and pan-CK mRNA, and then according to MKI67
Whether mRNA expression is positive to be classified as the TTF1 positive lung cancers CTCs of proliferation activity or the TTF1 positives without proliferation activity
Lung cancer CTCs.After each cell analysis finishes, the TTF1 positive lung cancers CTCs for collecting lung source in the blood sample is total
Number, and collect the TTF1 positive lung cancer CTCs numbers, percentage and the nothing in lung source that have proliferation activity in lung source respectively
The TTF1 positive lung cancer CTCs numbers of proliferation activity, percentage.
Gained testing result such as following table：
Table patients with lung cancer and normal control CTCs are counted and proliferation activity classification results
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, although with reference to foregoing reality
Apply example the present invention is described in detail, for those skilled in the art, it still can be to foregoing each implementation
Technical scheme described in example is modified, or carries out equivalent substitution to which part technical characteristic.All essences in the present invention
God any modification, equivalent substitution and improvements made etc., should be included in the scope of the protection with principle.
Claims (9)
1. a kind of lung cancer circulating tumor cell capture and activity test method, it is characterised in that：
Comprise the following steps：
(1) lung cancer CTCs capture：
1) peripheral blood is gathered：Routinely venous blood is adopted in operation, collects 5ml peripheric venous bloods；
2) crack and remove red blood cell：5ml peripheric venous bloods in step 1) are added in 45ml erythrocyte cracked liquids, overturned
The uninterrupted jog 20min of room temperature after mixing, with abundant splitting erythrocyte；500g centrifuges 10min, removes supernatant；Washed with cell
Cell precipitation is resuspended in liquid, and 1000g centrifuges 10min again, removes supernatant；
3) leucocyte is removed：Using magnetic bead combination leucocyte, cell precipitation is resuspended using 500 μ l cell washing solutions, CD45 is resisted
In body coating magnetic bead addition step 2) in cell suspension, it is put on rotary mixer and fully combines, 150r/min room temperature reactions
20min, after magnetic frame removes leucocyte, obtain the lung cancer CTCs cell suspensions for remaining a small amount of leucocyte；
4) miillpore filter retention lung cancer CTCs：The lung cancer CTCs cell suspensions obtained in step 3) are added into 50 μ L, 40% neutrality
After formalin solution fixes 8min, transfer cell suspension is taken out in filter of the aperture for 8 μm of miillpore filters by negative pressure
The less leukoreduction filter of diameter in cell suspension is fallen in suction, and the aperture that lung cancer CTCs is more than filter membrane because of diameter is trapped within filter membrane
On, lung cancer CTCs cells are obtained, it is standby；
(2) TTF1 positive lung cancers CTCs identification：
1) cell is fixed：It is molten that 1ml, 4% neutral formalin are added in the lung cancer CTCs cells that miillpore filter captures in step (1)
Liquid fills the fully fixed 1h of room temperature, and negative pressure leaching removes fixer, and after adding 1ml cell washing solutions washing 2min, suction filtration eliminates residual
Formaldehyde is stayed, the lung cancer CTCs cells after must fixing；
2) permeabilization：The filter membrane containing fixed lung cancer CTCs cells in above-mentioned steps is transferred in 24 orifice bores, adds 0.1%
Triton X-100 permeabilization agent, 5min is reacted at room temperature, increase membrane passage, cell washing solution is washed film 3 times, thoroughly gone
Except permeabilization agent, the lung cancer CTCs cells after permeabilization are obtained；
3) digest：Permeabilization lung cancer CTCs cells in above-mentioned steps are soaked in 2.5 μ g/ml pepsin working solutions, kept
40 DEG C of incubation 30min, wash film 3 times using cell washing solution, thoroughly remove pepsin, mRNA probes is entered cell
It is interior, obtain postdigestive lung cancer CTCs cells；
4) rna probe hybridization binding purpose mRNA molecules：Use three species specificity mRNA probes and mRNA points intracellular of target
Sub fully hybridization combines, and 40 DEG C of 2~3h of incubation, using the washing of probe cleaning solution three times, removes uncombined rna probe；
5) side chain DNA technique expands：On the basis of specific probe and target mRNA specific bonds, put using side chain DNA technique
Big hybridization signal, apply three kinds of different amplified fluorescence systems to three kinds of mRNA probe complexes respectively, finally cause every kind of
The peculiar fluorescence labeling of mRNA molecules is shown in cell in-situ；
6) nuclear targeting：Transfer is loaded with lung cancer CTCs miillpore filter and slide, adds DAPI fluorescent stainings liquid mark thin
Karyon, neutral gum mounting standby microscopy；
7) full-automatic fluorescence microscopy scarnning mirror and image is recorded：The 4 of each cell are scanned respectively using multichannel fluorescence microscope
Kind fluorescence signal, 4 kinds of fluorescence signals are respectively from DAPI (mark nucleus), Cy3 (mark TTF1 mRNA), FITC (marks
Pan-CK mRNA) and Cy5 (mark MKI67 mRNA) fluorescent dye, to lung cancer CTCs cells according to certain after the end of scan
Standard carries out activity analysis.
A kind of 2. activity assays of lung cancer circulating tumor cell, it is characterised in that：
Described TTF1 positive lung cancer CTCs Analysis of test results includes：To each cell captured from blood sample, successively
Analyze karyomorphism feature, three kinds of mRNA expressions of results；
First, standard is read to each cell to sort out：
(1) it is single by inspection three kinds of mRNA expression of results interpretation standards of cell：
Positive interpretation standard is expressed by inspection cell TTF1 mRNA：Fluorescence points >=3 under Cy3 passages；
Negative interpretation standard is expressed by inspection cell TTF1 mRNA：Fluorescence points ＜ 3 under Cy3 passages；
Positive interpretation standard is expressed by inspection cell pan-CK mRNA：Fluorescence points >=3 under FITC passages；
Negative interpretation standard is expressed by inspection cell pan-CK mRNA：Fluorescence points ＜ 3 under FITC passages；
Positive interpretation standard is expressed by inspection cell MKI67 mRNA：Fluorescence points >=3 under Cy5 passages；
Negative interpretation standard is expressed by inspection cell MKI67 mRNA：Fluorescence points ＜ 3 under Cy5 passages；
(2) TTF1 positive lung cancers CTCs interpretation standards：
For inspected each cell, if meeting three conditions in following criterion simultaneously, for TTF1 positive lung cancers
CTCs；
TTF1 positive lung cancer CTCs interpretation standards are：
1) cell nuclear diameter is more than neutrophil leucocyte and lymphocyte in leucocyte；
2) TTF1 mRNA are expressed as the positive；
3) pan-CK mRNA are expressed as the positive；
(3) TTF1 positive lung cancers CTCs proliferation activity interpretation standards：
There is the TTF1 positive lung cancers CTC of proliferation activity：On the premise of tri- interpretation conditions of TTF1 positive lung cancers CTCs are met,
MKI67 mRNA are expressed as the positive；
TTF1 positive lung cancers CTC without proliferation activity：On the premise of tri- interpretation conditions of TTF1 positive lung cancers CTCs are met,
MKI67 mRNA are expressed as feminine gender；
(4) testing result of each blood sample of analysis and summary：
According to above-mentioned interpretation standard, nucleus size is carried out to the doubtful CTCs captured from every blood sample and morphology is special
Sign analysis, differentiate whether be TTF1 positive lung cancer CTCs with reference to TTF1 mRNA and pan-CK mRNA, and then according to MKI67
Whether mRNA expression is positive to be classified as the TTF1 positive lung cancers CTCs of proliferation activity or the TTF1 positives without proliferation activity
Lung cancer CTCs.After each cell analysis finishes, the TTF1 positive lung cancers CTCs for collecting lung source in the blood sample is total
Number, and collect the TTF1 positive lung cancer CTCs numbers, percentage and the nothing in lung source that have proliferation activity in lung source respectively
The TTF1 positive lung cancer CTCs numbers of proliferation activity, percentage.
3. capture and the activity test method of lung cancer circulating tumor cell according to claim 1, it is characterised in that：It is described
MRNA probe combinations be：Pan-CK mRNA, TTF1 mRNA and MKI67 mRNA.
4. capture and the activity test method of lung cancer circulating tumor cell according to claim 1, it is characterised in that：It is described
Erythrocyte cracked liquid be：0.8% NH4Cl, 0.1% KHCO3With 0.1mM EDTA.
5. capture and the activity test method of lung cancer circulating tumor cell according to claim 1, it is characterised in that：It is described
Cell washing solution is：8.0g/L NaCl、0.2g/L KCl、1.44g/L Na2HPO4With 0.24g/L KH2PO4。
6. capture and the activity test method of lung cancer circulating tumor cell according to claim 1, it is characterised in that：It is described
Probe cleaning solution is：0.15M NaCl, 0.015M sodium citrates and 25% formamide.
7. a kind of capture of lung cancer circulating tumor cell and detection, analysis method, it is characterised in that：Using mRNA probe combinations and
Karyomorphism carries out activity classification to lung cancer CTCs.
8. a kind of capture of lung cancer circulating tumor cell and Activity determination, analysis method, it is characterised in that：Applied to patients with lung cancer
CTCs classification.
9. a kind of capture of lung cancer circulating tumor cell and Activity determination, analysis method, it is characterised in that：Applied to patients with lung cancer
Noninvasive auxiliary diagnosis.
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| CN113143328A (en) * | 2020-12-22 | 2021-07-23 | 上海市闵行区中心医院 | Noninvasive exfoliated gastric cell and nucleic acid sampling system and using method thereof |
| CN113143328B (en) * | 2020-12-22 | 2024-06-11 | 上海市闵行区中心医院 | Noninvasive gastric-shed cell and nucleic acid sampling system and application method thereof |
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