WO2018137515A1 - Immunofluorescent staining technique for urine exfoliated tumour cells of urothelial carcinoma - Google Patents

Immunofluorescent staining technique for urine exfoliated tumour cells of urothelial carcinoma Download PDF

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WO2018137515A1
WO2018137515A1 PCT/CN2018/072880 CN2018072880W WO2018137515A1 WO 2018137515 A1 WO2018137515 A1 WO 2018137515A1 CN 2018072880 W CN2018072880 W CN 2018072880W WO 2018137515 A1 WO2018137515 A1 WO 2018137515A1
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cell
protein
size
cd44v6
cell sorter
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PCT/CN2018/072880
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French (fr)
Chinese (zh)
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韩平畴
金百冶
陈安琪
傅广候
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瑞汉智芯医疗科技(嘉善)有限公司
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/021—Adjust spacings in an array of wells, pipettes or holders, format transfer between arrays of different size or geometry
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0893—Geometry, shape and general structure having a very large number of wells, microfabricated wells

Definitions

  • the invention belongs to the field of biological fluid external detection, and particularly relates to an immunofluorescence staining technique for urine exfoliated tumor cells of urothelial carcinoma.
  • Cytology is a commonly used method for detecting urothelial carcinoma in clinical practice. In order to find tumor cells, the method relies on the subjective observation of the smear by the pathologist (the cell is judged by factors such as the cell nucleus, chromatin, cytoplasm, etc.), and has the following disadvantages:
  • the detection sensitivity is low, generally only 30-50%.
  • Cystoscopy and ureteroscopy are the gold standard for clinical diagnosis of urothelial carcinoma.
  • the catheter is delivered to the target area through the urethra through a catheter to image and observe the suspected occupying position. But still has the following defects:
  • the tumor can only be observed when it is visible to the naked eye, which is not conducive to early diagnosis.
  • the filter membrane has a single diameter of 7.5 um; however, the urinary tract cancer has complex cellular components in the urine, in addition to tumor cells, normal detached urothelial cells, white blood cells, red blood cells, etc.
  • the cell size is inconsistent. If a single pore size is used, the cell components in the visual field will be confused and interfere with subsequent judgment and analysis;
  • an object of the present invention to provide an immunofluorescence staining technique for urinary exfoliated tumor cells of urothelial carcinoma.
  • a first aspect of the invention provides the use of a CK20 protein and a CD44v6 protein for the preparation or screening of a diagnostic reagent for urinary exfoliated tumor cells.
  • the CK20 protein and the CD44v6 protein are used in combination as a biomarker.
  • the CK20 protein and the CD44v6 protein are used together to prepare or screen the diagnostic reagent for urine exfoliated tumor cells, including two aspects.
  • the CK20 protein and the CD44v6 protein are used together to prepare a urine detachment tumor cell diagnostic reagent, which means The CK20 protein and CD44v6 protein were used together as diagnostic indicators for urine exfoliated tumor cells in the preparation of diagnostic reagents for urine exfoliated tumor cells.
  • the CK20 protein and the CD44v6 protein are collectively used as a standard or a positive control for the detection of urinary exfoliated tumor cells in a urine sample.
  • the CK20 protein and CD44v6 protein are used together to screen the diagnostic reagents for urinary exfoliated tumor cells.
  • the CK20 protein and CD44v6 protein are used together as a recognition target for urinary exfoliated tumor cells to screen for the specific recognition of CK20 protein and CD44v6 protein, respectively.
  • (including antibodies or ligands) as a diagnostic reagent for urinary exfoliated tumor cells.
  • antibodies that specifically bind to the CK20 protein and the CD44v6 protein, respectively are used to detect urinary exfoliated tumor cells in a urine sample.
  • antibodies that simultaneously bind specifically to the CK20 protein and the CD44v6 protein can also be used to detect urinary exfoliated tumor cells, such as bispecific antibodies, in urine samples, which are well known to those skilled in the art and will not be described again in detail.
  • the tumor is urothelial carcinoma.
  • the tumor is selected from the group consisting of, but not limited to, bladder cancer, ureteral cancer, and renal pelvic cancer.
  • a second aspect of the present invention provides the use of an agent which specifically or simultaneously recognizes a CK20 protein and a CD44v6 protein for the preparation of a urine detachment tumor cell diagnostic kit.
  • the agent that specifically or simultaneously recognizes the CK20 protein and the CD44v6 protein is selected from an antibody or ligand that specifically binds to the CK20 protein and the CD44v6 protein, respectively or simultaneously.
  • antibodies that specifically bind to the CK20 protein and the CD44v6 protein, respectively are used to detect urinary exfoliated tumor cells in a urine sample.
  • antibodies that simultaneously bind specifically to the CK20 protein and the CD44v6 protein can also be used to detect urinary exfoliated tumor cells, such as bispecific antibodies, in urine samples, which are well known to those skilled in the art and will not be described again in detail.
  • a third aspect of the present invention provides a urine detachment tumor cell diagnostic kit comprising at least an agent which specifically or simultaneously recognizes a CK20 protein and a CD44v6 protein, respectively.
  • the agent that specifically recognizes the CK20 protein and the CD44v6 protein is selected from an antibody or ligand that specifically binds to the CK20 protein and the CD44v6 protein.
  • antibodies that specifically bind to the CK20 protein and the CD44v6 protein, respectively are used to detect urinary exfoliated tumor cells in a urine sample.
  • antibodies that simultaneously bind specifically to the CK20 protein and the CD44v6 protein can also be used to detect urinary exfoliated tumor cells, such as bispecific antibodies, in urine samples, which are well known to those skilled in the art and will not be described again in detail.
  • the kit further comprises an immunologically binding (eg, antigen-antibody binding) reagent; or a fluorescent immunodetection reagent.
  • an immunologically binding (eg, antigen-antibody binding) reagent e.g, antigen-antibody binding
  • a fluorescent immunodetection reagent e.g., fluorescent immunodetection
  • the kit comprises: a first antibody that specifically binds to the CK20 protein, a first antibody that specifically binds to CD44v6, and a second antibody that is labeled with a fluorescein dye and specifically binds to the first antibody.
  • the kit further comprises a nuclear fluorescent dye.
  • CK20 protein, the CD44v6 protein, and the cells are positive at the same time, it can be determined that the urine is detached from the tumor cells.
  • the fluorescence wavelength emitted by the nuclear fluorescent dye after staining the nuclei is different from the fluorescence wavelength emitted by the two fluorescein dyes for labeling the second antibody, and can be distinguished.
  • it can be distinguished by a human eye or an optical image sensor (CCD, CMOS, etc.).
  • the fluorescein dye for labeling the second antibody is selected from, but not limited to, Alexa Fluor 488, Alexa 594, Cy3, Cy5.
  • the nuclear fluorescent dye is selected from, but not limited to, DAPI, Syto DNA, Hoechst 33342, PI (propidium iodide), EB (ethidium bromide).
  • the kit further comprises a microfluidic chip, the kit further comprising a microfluidic chip, the microfluidic chip is used for sorting and capturing urine exfoliated cells, and the microfluidic chip comprises sequentially connected a sample inlet, a cell capture region, and a sample outlet, the cell capture region being provided with a plurality of cell sorters, the cell sorter being composed of three columnar protrusions arranged in an overall arc shape, the columnar protrusions There is a gap between them, the arc-shaped opening serves as a liquid flow inlet, and the gap on both sides of the middle cylindrical protrusion serves as a liquid flow outlet, and the two liquid flow outlets are symmetrically distributed.
  • the cell capture zone is provided with three different size cell sorters, a first size cell sorter, a second size cell sorter and a third size cell sorter, respectively.
  • the size of the size cell sorter, the second size cell sorter, and the third size cell sorter are sequentially reduced.
  • adjacent cell sorters are different in size.
  • the microfluidic chip comprises a plurality of cell sorting units, each of the cell sorting units comprising a first size cell sorter, a second size cell sorter and a third size cell fraction The composition of the selector.
  • the cell sorters of the respective sizes in the cell sorting unit located in the same row are arranged in the same manner.
  • the laterally adjacent cell sorters have the same margins.
  • a first size cell sorter, a second size cell sorter and a third size cell sorter arranged in a horizontally equilaterally spaced arrangement form a first cell sorting unit; Arranging a first size cell sorter, a third size cell sorter and a second size cell sorter to form a second cell sorting unit; and the plurality of first cell sorting units are arranged in a lateral circulation arrangement a first cell sorting array, a plurality of second cell sorting units are laterally arranged to form a second cell sorting array, and a first cell sorting array and a second capturing array parallel to each other form a cell sorting stage
  • a plurality of mutually parallel cells in the cell capture region are sorted in a vertical equilateral marginal arrangement of the secondary array.
  • the midpoint of the flow inlet of the first size cell sorter of the second cell sorting array and the third size cell sorting of the corresponding first cell sorting array is aligned with the midpoint of the lateral margin of the second size cell sorter.
  • the first row of the first row of cell sorters of each odd row cell sorting secondary array is aligned longitudinally; each even row of cell sorting secondary arrays is adjacent to the previous odd row of cells sorting secondary array to the right Side isometric offset.
  • the flow inlet of the first size cell sorter has a width of 60 ⁇ 5 ⁇ m, the width of the liquid outlet is 22 ⁇ 5 ⁇ m, and the width of the liquid inlet of the second size cell sorter is 30 ⁇ 3 ⁇ m,
  • the width of the outflow port was 10 ⁇ 3 ⁇ m;
  • the width of the liquid inlet of the third-sized cell sorter was 16 ⁇ ⁇ 1 m, and the width of the liquid outlet was 4 ⁇ 1 ⁇ m.
  • the present invention has the following beneficial effects:
  • the present invention provides an efficient immunofluorescence staining technique for urinary exfoliating tumor cells of urothelial carcinoma.
  • two proteins of CK20 and CD44v6 were used as a group of markers, and the urine cells were determined by immunofluorescence to determine the tumor cells, and the detection accuracy of urine-shed tumor cells in human urine was improved, and the whole process was non-invasive, and Get rid of the dependence of conventional methods on the subjective experience of pathologists.
  • Figure 1 Schematic diagram of the structure and basic principle of the microfluidic chip.
  • Figure 2 Schematic diagram of the structure of the cell sorter.
  • Figure 3 Schematic diagram of cell sorting by different size cell sorters based on cell size differences.
  • Figure 4 Schematic diagram of the cell sorting unit in the cell capture zone of the microfluidic chip.
  • Figure 5 Schematic representation of a cell capture array in a cell capture zone of a microfluidic chip.
  • FIG. 6 Urine exfoliated cells captured by the microfluidic chip of the present invention, scale bar: 20 um.
  • Figure 7 Immunofluorescence images of urinary exfoliated tumor cells captured by microfluidic chips: DAPI+, CK20+, CD44v6+.
  • Figure 8 ROC curve obtained by microfluidic detection technique of urinary exfoliated cells in urothelial carcinoma.
  • Figure 9 Six urine exfoliated cells captured and recovered by the microfluidic chip of the present invention were subjected to single cell sequencing, and the CNV condition was analyzed.
  • CK20 protein and CD44v6 protein can be used together as a biomarker for diagnosing urinary exfoliated tumor cells: (i) differential diagnosis of urinary exfoliating tumor (urothelial carcinoma) cells, and / or susceptibility analysis; (ii) to assess the tumor (urothelial cancer) treatment of the relevant population, drug efficacy, patient prognosis, and the choice of appropriate treatment; (iii) to assess the relevant population of tumor (urothelial cancer) Disease risk, detection and early prevention and treatment.
  • the tumor is urothelial carcinoma.
  • the tumor is selected from the group consisting of, but not limited to, bladder cancer, ureteral cancer, and renal pelvic cancer.
  • the present invention provides a novel use of the CK20 protein and the CD44v6 protein for the preparation or screening of diagnostic reagents for urinary exfoliated tumor cells.
  • CK20 protein and CD44v6 protein are used together to prepare urinary exfoliating tumor cell diagnostic reagent, which refers to CK20 protein. It is used together with CD44v6 protein as a diagnostic indicator of urinary exfoliated tumor cells for the preparation of diagnostic reagents for urinary exfoliated tumor cells.
  • the CK20 protein and the CD44v6 protein are collectively used as a standard or a positive control for the detection of urinary exfoliated tumor cells in a urine sample.
  • the CK20 protein and CD44v6 protein are used together to screen the diagnostic reagents for urinary exfoliated tumor cells.
  • the CK20 protein and CD44v6 protein are used together as a recognition target for urinary exfoliated tumor cells to screen for the specific recognition of CK20 protein and CD44v6 protein, respectively.
  • (including antibodies or ligands) as a diagnostic reagent for urinary exfoliated tumor cells.
  • antibodies that specifically bind to the CK20 protein and the CD44v6 protein, respectively are used to detect urinary exfoliated tumor cells in a urine sample.
  • antibodies that simultaneously bind specifically to the CK20 protein and the CD44v6 protein can also be used to detect urinary exfoliated tumor cells, such as bispecific antibodies, in urine samples, which are well known to those skilled in the art and will not be described again in detail.
  • the tumor is urothelial carcinoma.
  • the tumor is selected from the group consisting of, but not limited to, bladder cancer, ureteral cancer, and renal pelvic cancer.
  • CK20 protein and CD44v6 protein are used together to prepare or screen the diagnostic reagents for urinary exfoliated tumor cells, reagents for specifically recognizing CK20 protein and CD44v6 protein, respectively or simultaneously, are used for preparing a urine detachment tumor cell diagnostic kit.
  • the agent that specifically or simultaneously recognizes the CK20 protein and the CD44v6 protein is selected from an antibody or a ligand that specifically binds to the CK20 protein and the CD44v6 protein, respectively or simultaneously.
  • antibodies that specifically bind to the CK20 protein and the CD44v6 protein, respectively are used to detect urinary exfoliated tumor cells in a urine sample.
  • antibodies that simultaneously bind specifically to the CK20 protein and the CD44v6 protein can also be used to detect urinary exfoliated tumor cells, such as bispecific antibodies, in urine samples, which are well known to those skilled in the art and will not be described again in detail.
  • CK20 protein and CD44v6 protein can be employed to detect the presence and expression of CK20 protein and CD44v6 protein on exfoliated cells, and such techniques are encompassed by the invention.
  • prior art techniques such as immunofluorescence staining, Southern blotting, Western blotting, and the like can be used, and these methods can be used in combination.
  • the present invention also provides an agent for detecting the presence or absence of CK20 protein and CD44v6 protein and expression in urine exfoliated cells.
  • an antibody that specifically recognizes the CK20 protein and the CD44v6 protein can be taken to determine the presence or absence of the CK20 protein and the CD44v6 protein.
  • Immunofluorescence staining can be used to detect the expression of CK20 protein and CD44v6 protein in urine exfoliated cells.
  • the detection reagent based on the immunofluorescence staining method may include a first antibody that specifically binds to the CK20 protein, a first antibody that specifically binds to CD44v6, a fluorescein dye-labeled second antibody that specifically binds to the first antibody, and a nucleus Fluorescent dyes.
  • the fluorescence wavelength emitted by the nuclear fluorescent dye after staining the nuclei is different from the fluorescence wavelength emitted by the two fluorescein dyes used to label the second antibody, and can be distinguished.
  • the fluorescein dye for labeling the second antibody is selected from, but not limited to, Alexa Fluor 488, Alexa 594, Cy3, Cy5.
  • the nuclear fluorescent dye is selected from, but not limited to, DAPI, Syto DNA, Hoechst 33342, PI (propidium iodide), EB (ethidium bromide).
  • Antibodies that bind to the CK20 protein and the CD44v6 protein can be prepared by various techniques known to those skilled in the art, and commercial antibodies can also be employed. For example, purified CK20 protein and CD44v6 protein can be administered to an animal to induce polyclonal antibody production. Similarly, cells expressing the CK20 protein and the CD44v6 protein can be used to immunize animals to produce antibodies. The antibodies found herein may also be monoclonal antibodies. Such cloned antibodies can be prepared using hybridoma technology (see Kohler et al, Nature 256; 495, 1975; Kohler et al, Eur. J. Immunol. 6: 511, 1976; Hammerling et al, In Monoclonal Antibodies and T Cell Hybridomas , Elsevier, NY, 1981).
  • Animals can be immunized with CK20 protein and CD44v6 protein, such as rabbits, mice, rats, and the like.
  • a variety of adjuvants can be used to enhance the immune response including, but not limited to, Freund's adjuvant and the like.
  • the first antibody that specifically binds to the CK20 protein is an Anti-Cytokeratin20 antibody (rabbit monoclonal antibody ab76126 from abcam), and the corresponding second antibody that specifically binds to it is Alexa Fluor. 488-labeled goat anti-rabbit IgG secondary antibody (Invitrogen, A11070).
  • the first antibody that specifically binds to CD44v6 is the Anti-CD44v6 antibody (a mouse monoclonal antibody ab78960 from abcam), and the corresponding second antibody that specifically binds to it is Alexa. 594-labeled goat anti-mouse IgG secondary antibody (abcam, ab150116).
  • the nuclear fluorescent dye is DAPI.
  • DAPI means 4,6 indole-2-phenylindole.
  • the affinity of DAPI for binding to DNA in the nucleus is extremely high. Therefore, the fluorescence emitted by DAPI mainly reflects the state of DNA distribution in the nucleus.
  • the kit further includes a microfluidic chip for sorting and capturing urine exfoliated cells.
  • the structure and principle of the microfluidic chip of the present invention can be seen in FIG. 1 .
  • the microfluidic chip includes a sample inlet 1-1, a cell capture region 1-2, and a sample outlet 1-3 that are sequentially connected in a liquid flow direction A.
  • the liquid sample to be treated, such as urine enters the microfluidic chip from the sample inlet 1-1
  • the cell capture zone 1-2 is the core region of the target cell for capture and sorting, and the target cell such as the cell capture zone 1-2
  • the urine exfoliated cells B are captured during the sorting process, and the remaining liquid flows out of the microfluidic chip from the sample outlets 1-3.
  • the cell trapping region 1-2 is provided with a plurality of cell sorters 2, as shown in Fig. 2, the cell sorter 2 is composed of three whole arcs.
  • the columnar protrusions 201 are arranged in a shape, and there is a gap between the columnar protrusions.
  • the arcuate opening 202 serves as a liquid flow inlet, and the gap 203 on both sides of the middle columnar protrusion serves as a liquid flow outlet, and the two liquid flow outlets are symmetrically distributed.
  • each cell sorter is curved to help maximize cell morphology integrity, and the design is an optimized design adapted to recover captured cells.
  • the buffer will flow from the reverse direction into the chip (flow from the sample outlet 1-3 to the sample inlet 1-1).
  • the cells will encounter many fronts.
  • Other cell sorters At this time, since the bottom of the cell sorter is curved, the cells are more easily bypassed by the cell sorter and are smoothly recovered, thereby greatly reducing the physical damage to the cells.
  • the capture region 1-2 is provided with three different size cell sorters 2, respectively being the first size cell sorter 2-1, Second size cell sorter 2-2 and third size cell sorter 2-3, first size cell sorter 2-1, second size cell sorter 2-2 and third size cell sorting
  • the size of the 2-3 is sequentially reduced.
  • Adjacent cell sorters are sized differently to create a turbulent flow environment throughout the cell capture zone 1-2. Therefore, target cells in a liquid sample, such as urinary exfoliated cells, randomly flow through cell sorters of different sizes, and when small-sized cells enter the large-sized cell sorter, they will be taken from the bottom of the large-sized cell sorter.
  • the two fluid outlets continue to advance as the flow continues until they encounter a matching cell sorter. Therefore, cells of different sizes are only captured by a cell sorter of a corresponding size, and target cells of different sizes such as urine exfoliated cells can be specifically captured. Small-sized cells do not occupy the space of large-sized cell sorters, greatly increasing the utilization of each cell sorter.
  • the width of the liquid inlet of the first size cell sorter 2-1 is 60 ⁇ 5 ⁇ m
  • the width of both liquid outlets is 22 ⁇ 5 ⁇ m
  • the diameter of the captureable stream is 22 ⁇ . Cells in the range of 60 ⁇ m.
  • the flow inlet of the second size cell sorter 2-2 has a width of 30 ⁇ 3 ⁇ m, and the widths of the two liquid flow outlets are all 10 ⁇ 3 ⁇ m, and cells having a diameter in the range of 10 to 22 ⁇ m can be captured.
  • the flow inlet of the third-sized cell sorter 2-3 has a width of 16 ⁇ 1 ⁇ m, and the widths of the two liquid flow outlets are all 4 ⁇ 1 ⁇ m, and cells having a diameter in the range of 4 to 10 ⁇ m can be captured.
  • the cells in the urine are generally larger than in the blood, and often there are cell clusters. Three different size cell sorters designed specifically for urine cells can avoid cell cluster defects.
  • the capture area 1-2 includes a plurality of cell sorting units 3, each of which is composed of a first size cell sorter 2-1 and a second size cell fraction.
  • the selector 2-2 is composed of a third size cell sorter 2-3.
  • the cell sorters of the respective sizes in the cell sorting unit located in the same row are arranged in the same manner.
  • the margin d1 between the laterally adjacent cell sorters is listed as 60 ⁇ 3 ⁇ m, which allows other impurities that may be present in the urine to pass smoothly, avoiding clogging of the chip. Even when there are a large number of cells in the urine of the patient and all the cell sorters in the microfluidic chip are occupied, the spacing of 60 ⁇ 3 ⁇ m allows excess cells to pass, thus avoiding the traditional filter method. The problem of confusion.
  • a first size cell sorter 2-1, a second size cell sorter 2-2 and a third size cell sorter 2-3 are arranged in a horizontally equilaterally spaced arrangement.
  • First cell sorting unit 3-1; a first size cell sorter 2-1, a third size cell sorter 2-3, and a second size cell sorter 2 arranged in a laterally equilaterally spaced arrangement -2 constitutes a second cell sorting unit 3-2;
  • a plurality of first cell sorting units 3-1 are arranged in a lateral circulation to constitute a first cell sorting array 4-1, and a plurality of second cell sorting units 3 - 2
  • the lateral circulation arrangement constitutes a second cell sorting array 4-2, a first cell sorting array and a second capturing array parallel to each other constitute a cell sorting secondary array 4, and a plurality of cells in the cell capturing region are parallel to each other
  • the cell sorting secondary array is arranged in a longitudinal equilateral margin.
  • the midpoint of the liquid inlet of the first size cell sorter 2-1 of the second cell sorting array 4-2 and the corresponding first cell sorting array is aligned with the midpoint of the lateral margin of the second 2-3 size cell sorter.
  • the longitudinal rows of the first row of the first row of the odd-row cell sorting secondary arrays are aligned in the longitudinal direction; the even-numbered rows of the cells are sorted into the secondary arrays and the adjacent odd-numbered rows of cells are sorted into the second array to the right.
  • Side isometric offset Helps create a turbulent, random flow environment that avoids cell orientation along a single path, thereby increasing the capture efficiency of microfluidic chips.
  • All target cells such as urine exfoliated cells flowing through the aforementioned microfluidic chip of the present invention will encounter the cell sorters of the respective stages with equal probability, thereby achieving the purpose of "collecting cells according to the difference in cell size.”
  • the observation field is more tidy and the cell morphology is clearer, which is conducive to the next step of analyzing the cell morphology (such as the current clinical urinary exfoliative cytology).
  • the microfluidic chip of the present invention can be prepared by a plasma treatment bond according to a standard process of polydimethylsiloxane (PDMS) using a conventional slide as a substrate.
  • PDMS polydimethylsiloxane
  • PDMS is the abbreviation for polydimethylsiloxane.
  • Polydimethylsiloxane is a curable polymer. After the curable polymer is mixed with a curing agent, it can be cured and hardened over a period of time to obtain a microfluidic chip with a certain structure.
  • microfluidic chip to sort and capture urine exfoliated cells is that (1) the cells captured by the microfluidic chip are distributed according to the size difference, so that the observation field is clear and convenient for the pathologist to read the film, thereby avoiding In the traditional membrane method, all the cells are squeezed together to interfere with the risk of subsequent reading; (2) When dyeing, the reagents used are controlled by a microfluidic injection pump, the dyeing time is accurate, and the dyeing process is automated.
  • the present invention utilizes a microfluidic chip to sort and capture urine exfoliated cells, and immunofluorescence method for detecting exfoliated tumor cells in human urine, which can accurately and objectively detect urothelial carcinoma (including bladder cancer, Ureteral cancer, renal pelvic cancer), and non-invasive throughout the process, and get rid of the dependence on the pathologist's subjective experience. Therefore, the invention effectively overcomes the problems of low sensitivity, subjective judgment, invasiveness and inefficiency of the pathologist according to the current clinical diagnosis of urothelial carcinoma.
  • urothelial carcinoma including bladder cancer, Ureteral cancer, renal pelvic cancer
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention employ molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields conventional in the art. Conventional technology. These techniques are well described in the existing literature. For details, see Sambrook et al.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons , New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman And AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, and the like.
  • the microfluidic chip of the present invention includes a sample inlet 1-1, a cell trapping region 1-2, and a sample outlet 1-3 which are sequentially connected in the liquid flow direction A.
  • the cell trapping area 1-2 is provided with a plurality of cell sorters 2, as shown in Fig. 2, the cell sorter 2 is composed of three columnar protrusions 201 arranged in an overall arc shape, between the columnar protrusions There is a gap, the arcuate opening 202 serves as a liquid flow inlet, and the gap 203 on both sides of the middle columnar projection serves as a liquid flow outlet, and the two liquid flow outlets are symmetrically distributed.
  • the cell capture zone 1-2 is provided with three different size cell sorters 2, a first size cell sorter 2-1, a second size cell sorter 2-2 and a third size cell sorter, respectively. 2-3, the sizes of the first size cell sorter 2-1, the second size cell sorter 2-2, and the third size cell sorter 2-3 are sequentially decreased.
  • the liquid inlet of the first size cell sorter 2-1 has a width of 60 ⁇ m, and the widths of the two liquid flow outlets are both 22 ⁇ m, and cells having a diameter in the range of 22 to 60 ⁇ m can be captured.
  • the flow inlet of the second-sized cell sorter 2-1 has a width of 30 ⁇ m, and both of the liquid outlets have a width of 10 ⁇ m, and cells having a diameter in the range of 10 to 22 ⁇ m can be captured.
  • the flow inlet of the third-sized cell sorter 2-3 has a width of 16 ⁇ m, and both of the liquid outlets have a width of 4 ⁇ m, and cells having a diameter in the range of 4 to 10 ⁇ m can be captured.
  • Adjacent cell sorters are sized differently to create a turbulent flow environment throughout the cell capture zone 1-2.
  • the capture area 1-2 includes a plurality of cell sorting units 3, each of which is composed of a first size cell sorter 2-1, a second size cell sorter 2-2, and A third size cell sorter consists of 2-3. Further, the cell sorters of the respective sizes in the cell sorting unit located in the same row are arranged in the same manner. The laterally adjacent cell sorters have the same margin d1 and are all 60 ⁇ m.
  • a first size cell sorter 2-1, a second size cell sorter 2-2 and a third size cell sorter 2-3 arranged in a horizontally equilaterally spaced arrangement form a first cell sorting unit 3-1; a first size cell sorter 2-1, a third size cell sorter 2-3, and a second size cell sorter 2-2 arranged side by side in a horizontally sequential manner
  • the cell sorting unit 3-2; the plurality of first cell sorting units 3-1 are arranged in a horizontal circulation to constitute the first cell sorting array 4-1, and the plurality of second cell sorting units 3-2 are arranged in a horizontal circulation arrangement.
  • the second cell sorting array 4-2, a first cell sorting array and a second capturing array parallel to each other constitute a cell sorting secondary array 4, and a plurality of mutually parallel cells in the cell capturing region are sorted into two
  • the rule is repeated until the microfluidic chip capture area 1-2 having a total length and a total width of 6000 ⁇ m is filled.
  • the microfluidic chip of the present invention was prepared by a plasma treatment bonding using a conventional 2.5*7.5 cm glass slide as a substrate according to a standard procedure of polydimethylsiloxane (PDMS).
  • PDMS polydimethylsiloxane
  • the urine sample to be treated or the PBS (phosphate buffer) suspension of urine sediment enters the microfluidic chip from the sample inlet 1-1, and the urine exfoliated cells pass through the cell capture zone 1-2. Captured, the remaining liquid exits the microfluidic chip from sample outlets 1-3.
  • the urine exfoliated cells B in the liquid sample are randomly flowed through the cell sorters of different sizes, and the urine exfoliated cells B of different sizes are captured by the cell sorter 2 of the corresponding size. Therefore, cells of different sizes are regularly distributed inside the chip, the field of view is clear, and the cell morphology is intact, which is particularly suitable for the next analysis.
  • Example 6 After the urine exfoliated cells are captured by the microfluidic chip in Example 1, as shown in Fig. 6, cells of different sizes are regularly distributed inside the chip, the visual field is clear, and the cell morphology is intact, which is particularly suitable for the next analysis.
  • urine detachment tumor cells and normal urothelial cells are distinguished by immunofluorescence.
  • Immunofluorescence staining step paraformaldehyde is passed through a microfluidic chip capturing urine exfoliated cells at a flow rate of 0.5-4 ml/h for 30 min; PBS is passed through the microfluidic chip at a flow rate of 0.5-4 ml/h to maintain 3 -5 min; a 0.1% Triton X-100 solution was passed through the microfluidic chip at a flow rate of 0.5 to 4 ml/h for 10 min; PBS was passed through the microfluidic chip at a flow rate of 0.5 to 4 ml/h. 3-5 min; BSA solution was passed through the microfluidic chip at a flow rate of 0.5 to 4 ml/h for 30 min.
  • Anti-CK20 primary antibody (abcam, ab76126), anti-CD44v6 primary antibody (abcam, ab78960) mixed solution was passed through the microfluidic chip at a flow rate of 0.5 ml/h for 60 min; PBS was flowed at a flow rate of 0.5-4 ml/h.
  • the microfluidic chip was maintained for 3-5 min; the CK20 corresponding secondary antibody (Invitrogen, A11070), CD44v6 corresponding secondary antibody (abcam, ab150116), DAPI (Invitrogen, MP01306) mixed solution passed through the micro at a flow rate of 0.5 ml/h.
  • the flow control chip was maintained for 30 min; the PBS was passed through the microfluidic chip at a flow rate of 0.5 to 4 ml/h for 3-5 min.
  • CK20 excitation wavelength 450 nm to 480 nm, emission wavelength 515 nm
  • CD44v6 excitation wavelength 515 nm to 585 nm, emission wavelength 610 nm
  • DAPI excitation wavelength 330 nm to 385 nm, emission wavelength 420 nm.
  • a major advantage of the present invention is that it can non-destructively capture and recover a single urinary exfoliated tumor cell, thereby laying the foundation for downstream single cell sequencing.
  • CNV copy number variation
  • the tumor cells showed a copy number disorder as expected, and the copy number of normal urothelial cells was consistent with the normal diploid characteristics.

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Abstract

Provided are an immunofluorescent staining reagent for urine exfoliated tumour cells of a urothelial carcinoma and an application thereof, comprising using the two proteins CK20 and CD44V6 as a set of markers and using an immunofluorescent method to identify urine exfoliated tumour cells in order to thereby detect a urothelial carcinoma.

Description

一种针对尿路上皮癌的尿脱落肿瘤细胞的免疫荧光染色技术Immunofluorescence staining technique for urine exfoliated tumor cells against urothelial carcinoma 技术领域Technical field
本发明属于生物体液体外检测领域，具体涉及一种针对尿路上皮癌的尿脱落肿瘤细胞的免疫荧光染色技术。The invention belongs to the field of biological fluid external detection, and particularly relates to an immunofluorescence staining technique for urine exfoliated tumor cells of urothelial carcinoma.
背景技术Background technique
1、临床现役技术-尿脱落细胞学检查：尿脱落细胞学检查(Cytology)是目前临床上普遍使用的尿路上皮癌检测方法。为寻找肿瘤细胞，该方法依赖病理医生对涂片进行主观观察(针对细胞的细胞核、染色质、胞浆等因素对细胞进行判断)，主要有以下缺点：1. Clinical active technology-urinary exfoliative cytology: Cytology is a commonly used method for detecting urothelial carcinoma in clinical practice. In order to find tumor cells, the method relies on the subjective observation of the smear by the pathologist (the cell is judged by factors such as the cell nucleus, chromatin, cytoplasm, etc.), and has the following disadvantages:
(1)检测敏感度低，一般只有30-50％。(1) The detection sensitivity is low, generally only 30-50%.
(2)阅片完全依赖于病理医生的主观经验，不同病理医生之间的判断结果或不一致。(2) Reading is completely dependent on the subjective experience of the pathologist, and the judgment results between different pathologists are inconsistent.
(3)染色过程繁琐，人工参与度大、工作强度大。细胞染色效果受人员经验、熟练度的影响。(3) The dyeing process is cumbersome, with large manual participation and high work intensity. Cell staining effects are influenced by personnel experience and proficiency.
2、临床现役技术-膀胱镜、输尿管软镜检查2, clinical active technology - cystoscopy, ureteroscopy
膀胱镜、输尿管软镜检查是目前临床诊断尿路上皮癌的金标准。其通过导管将窥镜经尿道传送至目标区域，对可疑占位进行成像、观察。但仍具有如下缺陷：Cystoscopy and ureteroscopy are the gold standard for clinical diagnosis of urothelial carcinoma. The catheter is delivered to the target area through the urethra through a catheter to image and observe the suspected occupying position. But still has the following defects:
(1)属于侵入式检查，患者所承受的痛苦大；(1) It is an invasive examination, and the suffering of the patient is large;
(2)肿瘤发展到肉眼可见时才能被观察到，不利于早期诊断.(2) The tumor can only be observed when it is visible to the naked eye, which is not conducive to early diagnosis.
3、其他在研技术-基于微滤膜的尿液细胞检查3, other research techniques - microfiltration membrane based urine cell examination
为克服传统尿脱落细胞学检查敏感度低的缺点，一些研究组使用微滤膜来检测尿脱落肿瘤细胞。相较于传统尿脱落细胞学检查，该方法利用滤膜过滤的原理捕获尿脱落细胞，敏感度略有提升，但仍具有以下缺陷：In order to overcome the shortcomings of traditional urine exfoliative cytology, some research groups used microfiltration membranes to detect urinary exfoliated tumor cells. Compared with the traditional urine exfoliative cytology, this method uses the principle of membrane filtration to capture urine exfoliated cells with a slight increase in sensitivity, but still has the following defects:
(1)捕获到细胞后，仍仅采用传统的病理学染色方法(巴氏染色)，该方法如同临床现役的尿脱落细胞学检查，高度依赖病理科医生，受主观因素的制约；(1) After the cells are captured, only the traditional pathological staining method (Pap Stain) is used. This method is like the clinically active urine exfoliative cytology, which is highly dependent on the pathologist and subject to subjective factors;
(2)该滤膜的滤孔口径单一，为7.5um；然而尿路上皮癌患者尿液中的细胞成分复杂，除了肿瘤细胞，还有正常脱落的尿路上皮细胞、白细胞、红细胞等，这些细胞尺寸均不一致，若使用单一孔径，仍将导致视野中细胞成分混乱，干扰后续的判断分析；(2) The filter membrane has a single diameter of 7.5 um; however, the urinary tract cancer has complex cellular components in the urine, in addition to tumor cells, normal detached urothelial cells, white blood cells, red blood cells, etc. The cell size is inconsistent. If a single pore size is used, the cell components in the visual field will be confused and interfere with subsequent judgment and analysis;
(3)无法对细胞的捕获过程进行实时成像、监测、调控，例如：(a)在细胞量过剩导致滤膜堵塞的情况下，无法及时终止进程；(b)在流速过高导致细胞破损严重的情况下，无 法实时控制流速；(3) It is impossible to perform real-time imaging, monitoring and regulation of the cell capture process, for example: (a) in the case of excess cell volume leading to clogging of the filter membrane, the process cannot be terminated in time; (b) the cell is damaged due to excessive flow rate The flow rate cannot be controlled in real time;
(4)受最大载荷量的限制，当细胞数量过高，所有滤孔均被占据,该滤膜随即失效；随后而来的细胞将全部堵塞于滤膜，导致视野混乱，难以进行下一步细胞层面的判读。(4) Limited by the maximum load, when the number of cells is too high, all the pores are occupied, and the filter will then fail; the subsequent cells will all block in the filter membrane, resulting in confusion of the field of view, making it difficult to proceed to the next step. Interpretation at the level.
发明内容Summary of the invention
为了克服现有技术中所存在的问题，本发明的目的在于提供一种针对尿路上皮癌的尿脱落肿瘤细胞的免疫荧光染色技术。In order to overcome the problems in the prior art, it is an object of the present invention to provide an immunofluorescence staining technique for urinary exfoliated tumor cells of urothelial carcinoma.
为了实现上述目的以及其他相关目的，本发明采用如下技术方案：In order to achieve the above and other related objects, the present invention adopts the following technical solutions:
本发明的第一方面提供了CK20蛋白和CD44v6蛋白共同用于制备或筛选尿脱落肿瘤细胞诊断试剂的用途。A first aspect of the invention provides the use of a CK20 protein and a CD44v6 protein for the preparation or screening of a diagnostic reagent for urinary exfoliated tumor cells.
优选地，CK20蛋白和CD44v6蛋白联用共同作为生物标志物。Preferably, the CK20 protein and the CD44v6 protein are used in combination as a biomarker.
优选地，CK20蛋白和CD44v6蛋白共同用于制备或筛选尿脱落肿瘤细胞诊断试剂的用途，包括两方面的内容，其一，CK20蛋白和CD44v6蛋白共同用于制备尿脱落肿瘤细胞诊断试剂，是指将CK20蛋白和CD44v6蛋白共同作为尿脱落肿瘤细胞的诊断指标应用于尿脱落肿瘤细胞诊断试剂的制备。在本发明一些实施方式中，列举了将CK20蛋白和CD44v6蛋白共同作为标准品或阳性对照，用于尿液样本中尿脱落肿瘤细胞的检测。Preferably, the CK20 protein and the CD44v6 protein are used together to prepare or screen the diagnostic reagent for urine exfoliated tumor cells, including two aspects. First, the CK20 protein and the CD44v6 protein are used together to prepare a urine detachment tumor cell diagnostic reagent, which means The CK20 protein and CD44v6 protein were used together as diagnostic indicators for urine exfoliated tumor cells in the preparation of diagnostic reagents for urine exfoliated tumor cells. In some embodiments of the invention, the CK20 protein and the CD44v6 protein are collectively used as a standard or a positive control for the detection of urinary exfoliated tumor cells in a urine sample.
其二，CK20蛋白和CD44v6蛋白共同用于筛选尿脱落肿瘤细胞诊断试剂，是指将CK20蛋白和CD44v6蛋白共同作为尿脱落肿瘤细胞的识别靶标筛选分别或同时特异性识别CK20蛋白和CD44v6蛋白的试剂(包括抗体或配体)，从而作为尿脱落肿瘤细胞诊断试剂。在本发明一些实施方式中，列举了用分别特异性结合CK20蛋白和CD44v6蛋白的抗体来检测尿液样本中尿脱落肿瘤细胞。此外，也可以采用同时特异性结合CK20蛋白和CD44v6蛋白的抗体来检测尿液样本中尿脱落肿瘤细胞，例如双特异性抗体，这些都是本领域技术人员所公知的技术，再次不在详述。Second, the CK20 protein and CD44v6 protein are used together to screen the diagnostic reagents for urinary exfoliated tumor cells. The CK20 protein and CD44v6 protein are used together as a recognition target for urinary exfoliated tumor cells to screen for the specific recognition of CK20 protein and CD44v6 protein, respectively. (including antibodies or ligands), as a diagnostic reagent for urinary exfoliated tumor cells. In some embodiments of the invention, antibodies that specifically bind to the CK20 protein and the CD44v6 protein, respectively, are used to detect urinary exfoliated tumor cells in a urine sample. In addition, antibodies that simultaneously bind specifically to the CK20 protein and the CD44v6 protein can also be used to detect urinary exfoliated tumor cells, such as bispecific antibodies, in urine samples, which are well known to those skilled in the art and will not be described again in detail.
优选地，所述肿瘤为尿路上皮癌。所述肿瘤选自但不限于膀胱癌、输尿管癌、肾盂癌。Preferably, the tumor is urothelial carcinoma. The tumor is selected from the group consisting of, but not limited to, bladder cancer, ureteral cancer, and renal pelvic cancer.
本发明的第二方面提供了分别或同时特异性识别CK20蛋白和CD44v6蛋白的试剂用于制备尿脱落肿瘤细胞诊断试剂盒的用途。A second aspect of the present invention provides the use of an agent which specifically or simultaneously recognizes a CK20 protein and a CD44v6 protein for the preparation of a urine detachment tumor cell diagnostic kit.
优选地，所述分别或同时特异性识别CK20蛋白和CD44v6蛋白的试剂选自分别或 同时特异性结合CK20蛋白和CD44v6蛋白的抗体或配体。Preferably, the agent that specifically or simultaneously recognizes the CK20 protein and the CD44v6 protein is selected from an antibody or ligand that specifically binds to the CK20 protein and the CD44v6 protein, respectively or simultaneously.
在本发明一些实施方式中，列举了用分别特异性结合CK20蛋白和CD44v6蛋白的抗体来检测尿液样本中尿脱落肿瘤细胞。此外，也可以采用同时特异性结合CK20蛋白和CD44v6蛋白的抗体来检测尿液样本中尿脱落肿瘤细胞，例如双特异性抗体，这些都是本领域技术人员所公知的技术，再次不在详述。In some embodiments of the invention, antibodies that specifically bind to the CK20 protein and the CD44v6 protein, respectively, are used to detect urinary exfoliated tumor cells in a urine sample. In addition, antibodies that simultaneously bind specifically to the CK20 protein and the CD44v6 protein can also be used to detect urinary exfoliated tumor cells, such as bispecific antibodies, in urine samples, which are well known to those skilled in the art and will not be described again in detail.
本发明的第三方面提供了一种尿脱落肿瘤细胞诊断试剂盒，所述的试剂盒中至少包括分别或同时特异性识别CK20蛋白和CD44v6蛋白的试剂。A third aspect of the present invention provides a urine detachment tumor cell diagnostic kit comprising at least an agent which specifically or simultaneously recognizes a CK20 protein and a CD44v6 protein, respectively.
优选地，所述特异性识别CK20蛋白和CD44v6蛋白的试剂选自特异性结合CK20蛋白和CD44v6蛋白的抗体或配体。在本发明一些实施方式中，列举了用分别特异性结合CK20蛋白和CD44v6蛋白的抗体来检测尿液样本中尿脱落肿瘤细胞。此外，也可以采用同时特异性结合CK20蛋白和CD44v6蛋白的抗体来检测尿液样本中尿脱落肿瘤细胞，例如双特异性抗体，这些都是本领域技术人员所公知的技术，再次不在详述。Preferably, the agent that specifically recognizes the CK20 protein and the CD44v6 protein is selected from an antibody or ligand that specifically binds to the CK20 protein and the CD44v6 protein. In some embodiments of the invention, antibodies that specifically bind to the CK20 protein and the CD44v6 protein, respectively, are used to detect urinary exfoliated tumor cells in a urine sample. In addition, antibodies that simultaneously bind specifically to the CK20 protein and the CD44v6 protein can also be used to detect urinary exfoliated tumor cells, such as bispecific antibodies, in urine samples, which are well known to those skilled in the art and will not be described again in detail.
优选地，所述试剂盒还包括免疫结合(如抗原抗体结合)试剂；或荧光免疫检测试剂。Preferably, the kit further comprises an immunologically binding (eg, antigen-antibody binding) reagent; or a fluorescent immunodetection reagent.
优选地，所述试剂盒中包括：特异性结合CK20蛋白的第一抗体，特异性结合CD44v6的第一抗体，以及荧光素染料标记的分别特异性结合所述第一抗体的第二抗体。Preferably, the kit comprises: a first antibody that specifically binds to the CK20 protein, a first antibody that specifically binds to CD44v6, and a second antibody that is labeled with a fluorescein dye and specifically binds to the first antibody.
优选地，所述试剂盒中还包括细胞核荧光染料。当CK20蛋白、CD44v6蛋白和细胞和三者同时阳性时，可判定为尿脱落肿瘤细胞。Preferably, the kit further comprises a nuclear fluorescent dye. When the CK20 protein, the CD44v6 protein, and the cells are positive at the same time, it can be determined that the urine is detached from the tumor cells.
进一步优选地，细胞核荧光染料对细胞核染色后发出的荧光波长与两种用于标记第二抗体的荧光素染料发出的荧光波长各不同，能够区别。例如，能够被人眼或光学图像传感器(CCD,CMOS等)区分。Further preferably, the fluorescence wavelength emitted by the nuclear fluorescent dye after staining the nuclei is different from the fluorescence wavelength emitted by the two fluorescein dyes for labeling the second antibody, and can be distinguished. For example, it can be distinguished by a human eye or an optical image sensor (CCD, CMOS, etc.).
所述用于标记第二抗体的荧光素染料选自但不限于Alexa Fluor 488、Alexa
Figure PCTCN2018072880-appb-000001
594、Cy3、Cy5。所述细胞核荧光染料选自但不限于：DAPI、Syto DNA、Hoechst33342、PI(碘化丙啶)、EB(溴化乙锭)。 The fluorescein dye for labeling the second antibody is selected from, but not limited to, Alexa Fluor 488, Alexa
Figure PCTCN2018072880-appb-000001
594, Cy3, Cy5. The nuclear fluorescent dye is selected from, but not limited to, DAPI, Syto DNA, Hoechst 33342, PI (propidium iodide), EB (ethidium bromide).
优选地，所述试剂盒还包括微流控芯片，所述试剂盒还包括微流控芯片，所述微流控芯片用于分选和捕获尿脱落细胞，所述微流控芯片包括依次连接的样本入口、细胞捕获区域以及样本出口，所述细胞捕获区域设有多个细胞分选器，所述细胞分选器由三个整体呈弧形排布的柱状凸起构成，柱状凸起之间存在间隙，弧形开口作为液流入口，中间柱状凸起两侧的间隙作为液流出口，两个液流出口呈对称分布。Preferably, the kit further comprises a microfluidic chip, the kit further comprising a microfluidic chip, the microfluidic chip is used for sorting and capturing urine exfoliated cells, and the microfluidic chip comprises sequentially connected a sample inlet, a cell capture region, and a sample outlet, the cell capture region being provided with a plurality of cell sorters, the cell sorter being composed of three columnar protrusions arranged in an overall arc shape, the columnar protrusions There is a gap between them, the arc-shaped opening serves as a liquid flow inlet, and the gap on both sides of the middle cylindrical protrusion serves as a liquid flow outlet, and the two liquid flow outlets are symmetrically distributed.
优选地，所述细胞捕获区域设有三种不同尺寸规格的细胞分选器，分别为第一尺寸细胞分选器、第二尺寸细胞分选器和第三尺寸细胞分选器，所述第一尺寸细胞分选器、 第二尺寸细胞分选器和第三尺寸细胞分选器的尺寸依次减小。Preferably, the cell capture zone is provided with three different size cell sorters, a first size cell sorter, a second size cell sorter and a third size cell sorter, respectively. The size of the size cell sorter, the second size cell sorter, and the third size cell sorter are sequentially reduced.
优选地，相邻的细胞分选器尺寸不同。Preferably, adjacent cell sorters are different in size.
优选地，所述微流控芯片包括多个细胞分选单元，每个所述细胞分选单元由一个第一尺寸细胞分选器、一个第二尺寸细胞分选器和一个第三尺寸细胞分选器组成。Preferably, the microfluidic chip comprises a plurality of cell sorting units, each of the cell sorting units comprising a first size cell sorter, a second size cell sorter and a third size cell fraction The composition of the selector.
优选地，位于同一行的细胞分选单元中各尺寸细胞分选器的排布方式相同。Preferably, the cell sorters of the respective sizes in the cell sorting unit located in the same row are arranged in the same manner.
优选地，横向相邻的细胞分选器，边距相同。Preferably, the laterally adjacent cell sorters have the same margins.
优选地，横向依次等边距排列的一个第一尺寸细胞分选器、一个第二尺寸细胞分选器和一个第三尺寸细胞分选器组成一个第一细胞分选单元；横向依次等边距排列的一个第一尺寸细胞分选器、一个第三尺寸细胞分选器和一个第二尺寸细胞分选器组成一个第二细胞分选单元；多个第一细胞分选单元横向循环排布构成第一细胞分选阵列，多个第二细胞分选单元横向循环排布构成第二细胞分选阵列，相互平行的一个第一细胞分选阵列和一个第二捕获阵列组成一个细胞分选二级阵列，细胞捕获区域中多个相互平行的细胞分选二级阵列纵向等边距循环排布。Preferably, a first size cell sorter, a second size cell sorter and a third size cell sorter arranged in a horizontally equilaterally spaced arrangement form a first cell sorting unit; Arranging a first size cell sorter, a third size cell sorter and a second size cell sorter to form a second cell sorting unit; and the plurality of first cell sorting units are arranged in a lateral circulation arrangement a first cell sorting array, a plurality of second cell sorting units are laterally arranged to form a second cell sorting array, and a first cell sorting array and a second capturing array parallel to each other form a cell sorting stage In the array, a plurality of mutually parallel cells in the cell capture region are sorted in a vertical equilateral marginal arrangement of the secondary array.
优选地，在每个细胞分选二级阵列中，第二细胞分选阵列的第一尺寸细胞分选器的液流入口的中点与对应第一细胞分选阵列的第三尺寸细胞分选器和第二尺寸细胞分选器横向边距的中点对齐。Preferably, in each of the cell sorting secondary arrays, the midpoint of the flow inlet of the first size cell sorter of the second cell sorting array and the third size cell sorting of the corresponding first cell sorting array The device is aligned with the midpoint of the lateral margin of the second size cell sorter.
优选地，各奇数行细胞分选二级阵列的首行首列细胞分选器纵向位置对齐；各偶数行细胞分选二级阵列较相邻的前一奇数行细胞分选二级阵列向右侧等距偏移。Preferably, the first row of the first row of cell sorters of each odd row cell sorting secondary array is aligned longitudinally; each even row of cell sorting secondary arrays is adjacent to the previous odd row of cells sorting secondary array to the right Side isometric offset.
优选地，第一尺寸细胞分选器的液流入口的宽度为60±5μm，液流出口的宽度为22±5μm；第二尺寸细胞分选器的液流入口的宽度为30±3μm，液流出口的宽度为10±3μm；第三尺寸细胞分选器的液流入口的宽度为16μ±1m，液流出口的宽度为4±1μm。Preferably, the flow inlet of the first size cell sorter has a width of 60±5 μm, the width of the liquid outlet is 22±5 μm, and the width of the liquid inlet of the second size cell sorter is 30±3 μm, The width of the outflow port was 10 ± 3 μm; the width of the liquid inlet of the third-sized cell sorter was 16 μ ± 1 m, and the width of the liquid outlet was 4 ± 1 μm.
与现有技术相比，本发明具有如下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
针对目前尿路上皮癌临床诊断中存在的敏感度低、依赖病理医生的主观判断、有创、低效等问题，本发明提供了一种高效的尿路上皮癌尿脱落肿瘤细胞免疫荧光染色技术，同时使用CK20和CD44v6两个蛋白作为一组标记物并采用免疫荧光法判定尿脱落肿瘤细胞来判定肿瘤细胞，提高了人尿液中的尿脱落肿瘤细胞的检测准确度，且全程无创，并摆脱了常规方法对于病理医生主观经验的依赖。In view of the low sensitivity of the current clinical diagnosis of urothelial carcinoma, subjective judgment, invasiveness, inefficiency and the like of the pathologist, the present invention provides an efficient immunofluorescence staining technique for urinary exfoliating tumor cells of urothelial carcinoma. At the same time, two proteins of CK20 and CD44v6 were used as a group of markers, and the urine cells were determined by immunofluorescence to determine the tumor cells, and the detection accuracy of urine-shed tumor cells in human urine was improved, and the whole process was non-invasive, and Get rid of the dependence of conventional methods on the subjective experience of pathologists.
附图说明DRAWINGS
图1：微流控芯片结构及基本原理示意图。Figure 1: Schematic diagram of the structure and basic principle of the microfluidic chip.
图2：细胞分选器的结构示意图。Figure 2: Schematic diagram of the structure of the cell sorter.
图3：不同尺寸的细胞分选器依据细胞尺寸差异分级捕获细胞原理示意图。Figure 3: Schematic diagram of cell sorting by different size cell sorters based on cell size differences.
图4：微流控芯片细胞捕获区中细胞分选单元示意图。Figure 4: Schematic diagram of the cell sorting unit in the cell capture zone of the microfluidic chip.
图5：微流控芯片细胞捕获区中细胞捕获阵列示意图。Figure 5: Schematic representation of a cell capture array in a cell capture zone of a microfluidic chip.
图6：本发明微流控芯片捕获到的尿脱落细胞，比例尺：20um。Figure 6: Urine exfoliated cells captured by the microfluidic chip of the present invention, scale bar: 20 um.
图7：微流控芯片捕获到的尿脱落肿瘤细胞的免疫荧光图像：DAPI+、CK20+、CD44v6+。Figure 7: Immunofluorescence images of urinary exfoliated tumor cells captured by microfluidic chips: DAPI+, CK20+, CD44v6+.
图8：尿路上皮癌尿脱落细胞微流检测技术所得的ROC曲线。Figure 8: ROC curve obtained by microfluidic detection technique of urinary exfoliated cells in urothelial carcinoma.
图9：利用本发明微流控芯片捕获、回收的6个尿脱落细胞进行单细胞测序，并分析其CNV情况。Figure 9: Six urine exfoliated cells captured and recovered by the microfluidic chip of the present invention were subjected to single cell sequencing, and the CNV condition was analyzed.
元件标号说明Component label description
1-1                    样本入口1-1 sample entrance
1-2                    细胞捕获区域1-2 cell capture area
1-3                    样本出口1-3 sample export
2                      细胞分选器2 cell sorter
201                    柱状凸起201 columnar projection
202                    液流入口202 liquid inlet
203                    液流出口203 liquid outlet
2-1                    第一尺寸细胞分选器2-1 first size cell sorter
2-2                    第二尺寸细胞分选器2-2 second size cell sorter
2-3                    第三尺寸细胞分选器2-3 third size cell sorter
3                      细胞分选单元3 cell sorting unit
3-1                    第一细胞分选单元3-1 First cell sorting unit
3-2                    第二细胞分选单元3-2 second cell sorting unit
4                      细胞分选二级阵列4 cell sorting secondary array
4-1                   第一细胞分选阵列4-1 First Cell Sorting Array
4-2                   第二细胞分选阵列4-2 second cell sorting array
具体实施方式detailed description
一、CK20蛋白和CD44v6蛋白的用途First, the use of CK20 protein and CD44v6 protein
本发明经过广泛而深入的研究，首次发现CK20蛋白和CD44v6蛋白可联用共同作为诊断尿脱落肿瘤细胞的生物标志物：(i)进行尿脱落肿瘤(尿路上皮癌)细胞的鉴别诊断，和/或易感分析；(ii)评估相关人群的肿瘤(尿路上皮癌)治疗药物、药物疗效、患者预后，以及选择合适的治疗方法；(iii)评估相关人群肿瘤(尿路上皮癌)患病风险、检测并进行早期防治。The invention has been extensively and deeply studied, and it is first discovered that CK20 protein and CD44v6 protein can be used together as a biomarker for diagnosing urinary exfoliated tumor cells: (i) differential diagnosis of urinary exfoliating tumor (urothelial carcinoma) cells, and / or susceptibility analysis; (ii) to assess the tumor (urothelial cancer) treatment of the relevant population, drug efficacy, patient prognosis, and the choice of appropriate treatment; (iii) to assess the relevant population of tumor (urothelial cancer) Disease risk, detection and early prevention and treatment.
进一步地，所述肿瘤为尿路上皮癌。所述肿瘤选自但不限于膀胱癌、输尿管癌、肾盂癌。Further, the tumor is urothelial carcinoma. The tumor is selected from the group consisting of, but not limited to, bladder cancer, ureteral cancer, and renal pelvic cancer.
因此，本发明提供了CK20蛋白和CD44v6蛋白共同用于制备或筛选尿脱落肿瘤细胞诊断试剂的新用途。Accordingly, the present invention provides a novel use of the CK20 protein and the CD44v6 protein for the preparation or screening of diagnostic reagents for urinary exfoliated tumor cells.
CK20蛋白和CD44v6蛋白共同用于制备或筛选尿脱落肿瘤细胞诊断试剂的用途，包括两方面的内容，其一，CK20蛋白和CD44v6蛋白共同用于制备尿脱落肿瘤细胞诊断试剂，是指将CK20蛋白和CD44v6蛋白共同作为尿脱落肿瘤细胞的诊断指标应用于尿脱落肿瘤细胞诊断试剂的制备。在本发明一些实施方式中，列举了将CK20蛋白和CD44v6蛋白共同作为标准品或阳性对照，用于尿液样本中尿脱落肿瘤细胞的检测。其二，CK20蛋白和CD44v6蛋白共同用于筛选尿脱落肿瘤细胞诊断试剂，是指将CK20蛋白和CD44v6蛋白共同作为尿脱落肿瘤细胞的识别靶标筛选分别或同时特异性识别CK20蛋白和CD44v6蛋白的试剂(包括抗体或配体)，从而作为尿脱落肿瘤细胞诊断试剂。在本发明一些实施方式中，列举了用分别特异性结合CK20蛋白和CD44v6蛋白的抗体来检测尿液样本中尿脱落肿瘤细胞。此外，也可以采用同时特异性结合CK20蛋白和CD44v6蛋白的抗体来检测尿液样本中尿脱落肿瘤细胞，例如双特异性抗体，这些都是本领域技术人员所公知的技术，再次不在详述。The use of CK20 protein and CD44v6 protein for the preparation or screening of urinary exfoliative tumor cell diagnostic reagents includes two aspects. First, CK20 protein and CD44v6 protein are used together to prepare urinary exfoliating tumor cell diagnostic reagent, which refers to CK20 protein. It is used together with CD44v6 protein as a diagnostic indicator of urinary exfoliated tumor cells for the preparation of diagnostic reagents for urinary exfoliated tumor cells. In some embodiments of the invention, the CK20 protein and the CD44v6 protein are collectively used as a standard or a positive control for the detection of urinary exfoliated tumor cells in a urine sample. Second, the CK20 protein and CD44v6 protein are used together to screen the diagnostic reagents for urinary exfoliated tumor cells. The CK20 protein and CD44v6 protein are used together as a recognition target for urinary exfoliated tumor cells to screen for the specific recognition of CK20 protein and CD44v6 protein, respectively. (including antibodies or ligands), as a diagnostic reagent for urinary exfoliated tumor cells. In some embodiments of the invention, antibodies that specifically bind to the CK20 protein and the CD44v6 protein, respectively, are used to detect urinary exfoliated tumor cells in a urine sample. In addition, antibodies that simultaneously bind specifically to the CK20 protein and the CD44v6 protein can also be used to detect urinary exfoliated tumor cells, such as bispecific antibodies, in urine samples, which are well known to those skilled in the art and will not be described again in detail.
所述肿瘤为尿路上皮癌。所述肿瘤选自但不限于膀胱癌、输尿管癌、肾盂癌。The tumor is urothelial carcinoma. The tumor is selected from the group consisting of, but not limited to, bladder cancer, ureteral cancer, and renal pelvic cancer.
二、分别或同时特异性识别CK20蛋白和CD44v6蛋白的试剂的用途2. Use of reagents for specifically and simultaneously recognizing CK20 protein and CD44v6 protein
基于CK20蛋白和CD44v6蛋白共同用于制备或筛选尿脱落肿瘤细胞诊断试剂的发现，分别或同时特异性识别CK20蛋白和CD44v6蛋白的试剂用于制备尿脱落肿瘤细胞诊断试剂盒。Based on the discovery that CK20 protein and CD44v6 protein are used together to prepare or screen the diagnostic reagents for urinary exfoliated tumor cells, reagents for specifically recognizing CK20 protein and CD44v6 protein, respectively or simultaneously, are used for preparing a urine detachment tumor cell diagnostic kit.
进一步地，所述分别或同时特异性识别CK20蛋白和CD44v6蛋白的试剂选自分别或同时特异性结合CK20蛋白和CD44v6蛋白的抗体或配体。Further, the agent that specifically or simultaneously recognizes the CK20 protein and the CD44v6 protein is selected from an antibody or a ligand that specifically binds to the CK20 protein and the CD44v6 protein, respectively or simultaneously.
在本发明一些实施方式中，列举了用分别特异性结合CK20蛋白和CD44v6蛋白的抗体来检测尿液样本中尿脱落肿瘤细胞。此外，也可以采用同时特异性结合CK20蛋白和CD44v6蛋白的抗体来检测尿液样本中尿脱落肿瘤细胞，例如双特异性抗体，这些都是本领域技术人员所公知的技术，再次不在详述。In some embodiments of the invention, antibodies that specifically bind to the CK20 protein and the CD44v6 protein, respectively, are used to detect urinary exfoliated tumor cells in a urine sample. In addition, antibodies that simultaneously bind specifically to the CK20 protein and the CD44v6 protein can also be used to detect urinary exfoliated tumor cells, such as bispecific antibodies, in urine samples, which are well known to those skilled in the art and will not be described again in detail.
三、尿脱落肿瘤细胞诊断试剂盒Third, urine detachment tumor cell diagnostic kit
可采用各种本领域已知的技术来检测尿脱落细胞上CK20蛋白和CD44v6蛋白的存在与否及表达情况，这些技术均包含在发明中。例如，可用已有技术如免疫荧光染色法、Southern印迹法、Wester印迹法等，这些方法可结合使用。A variety of techniques known in the art can be employed to detect the presence and expression of CK20 protein and CD44v6 protein on exfoliated cells, and such techniques are encompassed by the invention. For example, prior art techniques such as immunofluorescence staining, Southern blotting, Western blotting, and the like can be used, and these methods can be used in combination.
本发明还提供用于在尿脱落细胞中检测CK20蛋白和CD44v6蛋白的存在与否以及表达情况的试剂。优选地，当进行蛋白存在与否以及表达情况的检测时，可采取特异性识别CK20蛋白和CD44v6蛋白的抗体来确定CK20蛋白和CD44v6蛋白的存在与否。The present invention also provides an agent for detecting the presence or absence of CK20 protein and CD44v6 protein and expression in urine exfoliated cells. Preferably, when the presence or absence of the protein and the detection of the expression are performed, an antibody that specifically recognizes the CK20 protein and the CD44v6 protein can be taken to determine the presence or absence of the CK20 protein and the CD44v6 protein.
利用特异性结合CK20蛋白和CD44v6蛋白的抗体来检测尿脱落细胞中CK20蛋白和CD44v6蛋白表达情况的方法也是本领域人员熟知的技术。Methods for detecting expression of CK20 protein and CD44v6 protein in urinary exfoliated cells using antibodies that specifically bind to CK20 protein and CD44v6 protein are also well known in the art.
可采用免疫荧光染色的方法来检测尿脱落细胞中CK20蛋白和CD44v6蛋白表达情况。基于免疫荧光染色方法的检测试剂可包括特异性结合CK20蛋白的第一抗体，特异性结合CD44v6的第一抗体，分别荧光素染料标记的特异性结合所述第一抗体的第二抗体，以及细胞核荧光染料。细胞核荧光染料对细胞核染色后发出的荧光波长与两种用于标记第二抗体的荧光素染料发出的荧光波长不同，能够区别。例如，能够被人眼或光学图像传感器(CCD,CMOS等)区分。所述用于标记第二抗体的荧光素染料选自但不限于Alexa Fluor488、Alexa
Figure PCTCN2018072880-appb-000002
594、Cy3、Cy5。所述细胞核荧光染料选自但不限于：DAPI、Syto DNA、Hoechst33342、PI(碘化丙啶)、EB(溴化乙锭)。 Immunofluorescence staining can be used to detect the expression of CK20 protein and CD44v6 protein in urine exfoliated cells. The detection reagent based on the immunofluorescence staining method may include a first antibody that specifically binds to the CK20 protein, a first antibody that specifically binds to CD44v6, a fluorescein dye-labeled second antibody that specifically binds to the first antibody, and a nucleus Fluorescent dyes. The fluorescence wavelength emitted by the nuclear fluorescent dye after staining the nuclei is different from the fluorescence wavelength emitted by the two fluorescein dyes used to label the second antibody, and can be distinguished. For example, it can be distinguished by a human eye or an optical image sensor (CCD, CMOS, etc.). The fluorescein dye for labeling the second antibody is selected from, but not limited to, Alexa Fluor 488, Alexa
Figure PCTCN2018072880-appb-000002
594, Cy3, Cy5. The nuclear fluorescent dye is selected from, but not limited to, DAPI, Syto DNA, Hoechst 33342, PI (propidium iodide), EB (ethidium bromide).
可以通过本领域内技术人员已知的各种技术制备结合CK20蛋白和CD44v6蛋白的抗体，也可采用商业化的抗体。例如，纯化的CK20蛋白和CD44v6蛋白，可被施用于动物以诱导多克隆抗体产生。与之相似的，表达CK20蛋白和CD44v6蛋白的细胞可用来免疫动物 来产生抗体。本发现的抗体也可以是单克隆抗体。此类克隆抗体可以利用杂交瘤技术来制备(见Kohler等人，Nature256；495,1975；Kohler等人，Eur.J.Immunol.6:511,1976；Hammerling等人，In Monoclonal Antibodies and T Cell Hybridomas，Elsevier，N.Y.，1981)。Antibodies that bind to the CK20 protein and the CD44v6 protein can be prepared by various techniques known to those skilled in the art, and commercial antibodies can also be employed. For example, purified CK20 protein and CD44v6 protein can be administered to an animal to induce polyclonal antibody production. Similarly, cells expressing the CK20 protein and the CD44v6 protein can be used to immunize animals to produce antibodies. The antibodies found herein may also be monoclonal antibodies. Such cloned antibodies can be prepared using hybridoma technology (see Kohler et al, Nature 256; 495, 1975; Kohler et al, Eur. J. Immunol. 6: 511, 1976; Hammerling et al, In Monoclonal Antibodies and T Cell Hybridomas , Elsevier, NY, 1981).
多克隆抗体的生产可用CK20蛋白和CD44v6蛋白免疫动物，如家兔、小鼠、大鼠等。多种佐剂可用于增强免疫反应，包括但不限于弗氏佐剂等。Production of polyclonal antibodies Animals can be immunized with CK20 protein and CD44v6 protein, such as rabbits, mice, rats, and the like. A variety of adjuvants can be used to enhance the immune response including, but not limited to, Freund's adjuvant and the like.
本发明的一些实施方式中列举了：特异性结合CK20蛋白的第一抗体为Anti-Cytokeratin20抗体(购自abcam的兔单克隆抗体ab76126)，对应的与之特异性结合的第二抗体为Alexa Fluor 488标记的羊抗兔IgG二抗(Invitrogen,A11070)。特异性结合CD44v6的第一抗体为Anti-CD44v6抗体(购自abcam的小鼠单克隆抗体ab78960)，对应的与之特异性结合的第二抗体为Alexa
Figure PCTCN2018072880-appb-000003
594标记的山羊抗小鼠IgG二抗(abcam,ab150116)。细胞核荧光染料为DAPI。DAPI是指4,6联脒-2-苯基吲哚。DAPI与细胞核内的DNA结合的亲和力极高。因此，DAPI发射的荧光主要反映了细胞核内的DNA分布状态。 In some embodiments of the present invention, the first antibody that specifically binds to the CK20 protein is an Anti-Cytokeratin20 antibody (rabbit monoclonal antibody ab76126 from abcam), and the corresponding second antibody that specifically binds to it is Alexa Fluor. 488-labeled goat anti-rabbit IgG secondary antibody (Invitrogen, A11070). The first antibody that specifically binds to CD44v6 is the Anti-CD44v6 antibody (a mouse monoclonal antibody ab78960 from abcam), and the corresponding second antibody that specifically binds to it is Alexa.
Figure PCTCN2018072880-appb-000003
594-labeled goat anti-mouse IgG secondary antibody (abcam, ab150116). The nuclear fluorescent dye is DAPI. DAPI means 4,6 indole-2-phenylindole. The affinity of DAPI for binding to DNA in the nucleus is extremely high. Therefore, the fluorescence emitted by DAPI mainly reflects the state of DNA distribution in the nucleus.
进一步地，所述试剂盒还包括微流控芯片，所述微流控芯片用于分选和捕获尿脱落细胞，本发明的微流控芯片的结构及原理示意图可参见图1。具体地，所述微流控芯片，包括沿液流方向A依次连接的样本入口1-1、细胞捕获区域1-2以及样本出口1-3。待处理的液体样本如尿液，从样本入口1-1进入微流控芯片，细胞捕获区域1-2是目标细胞的捕获与分选的核心区域，途径细胞捕获区域1-2时目标细胞如尿脱落细胞B在分选的过程中被捕获，剩余液体从样本出口1-3流出微流控芯片。Further, the kit further includes a microfluidic chip for sorting and capturing urine exfoliated cells. The structure and principle of the microfluidic chip of the present invention can be seen in FIG. 1 . Specifically, the microfluidic chip includes a sample inlet 1-1, a cell capture region 1-2, and a sample outlet 1-3 that are sequentially connected in a liquid flow direction A. The liquid sample to be treated, such as urine, enters the microfluidic chip from the sample inlet 1-1, the cell capture zone 1-2 is the core region of the target cell for capture and sorting, and the target cell such as the cell capture zone 1-2 The urine exfoliated cells B are captured during the sorting process, and the remaining liquid flows out of the microfluidic chip from the sample outlets 1-3.
为了适用于目标细胞如尿脱落细胞的分选与捕获，细胞捕获区域1-2设有多个细胞分选器2，如图2所示，所述细胞分选器2由三个整体呈弧形排布的柱状凸起201构成，柱状凸起之间存在间隙，弧形开口202作为液流入口，中间柱状凸起两侧的间隙203作为液流出口，两个液流出口呈对称分布。In order to be suitable for sorting and capturing of target cells such as urine exfoliated cells, the cell trapping region 1-2 is provided with a plurality of cell sorters 2, as shown in Fig. 2, the cell sorter 2 is composed of three whole arcs. The columnar protrusions 201 are arranged in a shape, and there is a gap between the columnar protrusions. The arcuate opening 202 serves as a liquid flow inlet, and the gap 203 on both sides of the middle columnar protrusion serves as a liquid flow outlet, and the two liquid flow outlets are symmetrically distributed.
进一步地，各细胞分选器的底部呈弧状，有助于最大程度保持细胞形态完整，该设计是一种适应于回收被捕获细胞的优化设计。在回收所捕获的细胞时，缓冲液会从反向流入芯片(从样本出口1-3方向流向样本入口1-1方向)，当细胞被冲出原细胞分选器时，将遇到前方众多的其他细胞分选器。此时，因细胞分选器的底部为弧形，细胞便更容易绕过细胞分选器而被顺利回收，从而大大降低了细胞受到的物理损伤。Further, the bottom of each cell sorter is curved to help maximize cell morphology integrity, and the design is an optimized design adapted to recover captured cells. When recovering the captured cells, the buffer will flow from the reverse direction into the chip (flow from the sample outlet 1-3 to the sample inlet 1-1). When the cells are flushed out of the original cell sorter, they will encounter many fronts. Other cell sorters. At this time, since the bottom of the cell sorter is curved, the cells are more easily bypassed by the cell sorter and are smoothly recovered, thereby greatly reducing the physical damage to the cells.
进一步地，如图3所示，为了依据细胞尺寸差异分级捕获细胞细胞，捕获区域1-2设有三种不同尺寸规格的细胞分选器2，分别为第一尺寸细胞分选器2-1、第二尺寸细胞分选器2-2和第三尺寸细胞分选器2-3，第一尺寸细胞分选器2-1、第二尺寸细胞分选器2-2和第三 尺寸细胞分选器2-3的尺寸依次减小。相邻的细胞分选器尺寸不同，在整个细胞捕获区域1-2内营造紊乱的液流环境。因此，液体样本中的目标细胞，如尿脱落细胞随机流经各个不同尺寸规格的细胞分选器，当小尺寸的细胞进入大尺寸细胞分选器后，会从大尺寸细胞分选器底部的两个液流出口，并随着液流继续往前行，直到遇到尺寸相匹配的细胞分选器而被捕获。所以，不同尺寸的细胞只被对应尺寸的细胞分选器捕获，可针对性地捕获不同大小的目标细胞如尿脱落细胞。小尺寸细胞不会占据大尺寸细胞分选器的空间，大幅提高了各细胞分选器的利用率。本发明的一些实施方式中列举了：第一尺寸细胞分选器2-1的液流入口的宽度为60±5μm，两个液流出口的宽度均为22±5μm，可捕获直径在22～60μm范围内的细胞。第二尺寸细胞分选器2-2的液流入口的宽度为30±3μm，两个液流出口的宽度均为10±3μm，可捕获直径在10～22μm范围内的细胞。第三尺寸细胞分选器2-3的液流入口的宽度为16±1μm，两个液流出口的宽度均为4±1μm，可捕获直径在4～10μm范围内的细胞。尿液中的细胞普遍比血液中大，且常常存在细胞团的情况，针对尿液细胞特定设计的三种不同尺寸规格的细胞分选器，可避免出现细胞团的缺陷。Further, as shown in FIG. 3, in order to hierarchically capture the cell cells according to the difference in cell size, the capture region 1-2 is provided with three different size cell sorters 2, respectively being the first size cell sorter 2-1, Second size cell sorter 2-2 and third size cell sorter 2-3, first size cell sorter 2-1, second size cell sorter 2-2 and third size cell sorting The size of the 2-3 is sequentially reduced. Adjacent cell sorters are sized differently to create a turbulent flow environment throughout the cell capture zone 1-2. Therefore, target cells in a liquid sample, such as urinary exfoliated cells, randomly flow through cell sorters of different sizes, and when small-sized cells enter the large-sized cell sorter, they will be taken from the bottom of the large-sized cell sorter. The two fluid outlets continue to advance as the flow continues until they encounter a matching cell sorter. Therefore, cells of different sizes are only captured by a cell sorter of a corresponding size, and target cells of different sizes such as urine exfoliated cells can be specifically captured. Small-sized cells do not occupy the space of large-sized cell sorters, greatly increasing the utilization of each cell sorter. In some embodiments of the present invention, the width of the liquid inlet of the first size cell sorter 2-1 is 60±5 μm, the width of both liquid outlets is 22±5 μm, and the diameter of the captureable stream is 22～. Cells in the range of 60 μm. The flow inlet of the second size cell sorter 2-2 has a width of 30 ± 3 μm, and the widths of the two liquid flow outlets are all 10 ± 3 μm, and cells having a diameter in the range of 10 to 22 μm can be captured. The flow inlet of the third-sized cell sorter 2-3 has a width of 16 ± 1 μm, and the widths of the two liquid flow outlets are all 4 ± 1 μm, and cells having a diameter in the range of 4 to 10 μm can be captured. The cells in the urine are generally larger than in the blood, and often there are cell clusters. Three different size cell sorters designed specifically for urine cells can avoid cell cluster defects.
如图4所示，所述捕获区域1-2包括多个细胞分选单元3，每个所述细胞分选单元3由一个第一尺寸细胞分选器2-1、一个第二尺寸细胞分选器2-2和一个第三尺寸细胞分选器2-3组成。进一步地，位于同一行的细胞分选单元中各尺寸细胞分选器的排布方式相同。横向相邻的细胞分选器，边距d1相同。本发明的一些实施例中，列举了横向相邻的细胞分选器之间的边距d1为60±3μm，该尺寸允许尿液中可能出现的其他杂质顺利通过，避免了芯片的堵塞。即便是当患者尿液中有大量的细胞而将微流控芯片内所有细胞分选器均已被占据时，60±3μm的间距也能允许过剩的细胞通过，从而避免了传统滤膜法中视野混乱的问题。As shown in FIG. 4, the capture area 1-2 includes a plurality of cell sorting units 3, each of which is composed of a first size cell sorter 2-1 and a second size cell fraction. The selector 2-2 is composed of a third size cell sorter 2-3. Further, the cell sorters of the respective sizes in the cell sorting unit located in the same row are arranged in the same manner. Horizontally adjacent cell sorters with the same margin d1. In some embodiments of the invention, the margin d1 between the laterally adjacent cell sorters is listed as 60 ± 3 μm, which allows other impurities that may be present in the urine to pass smoothly, avoiding clogging of the chip. Even when there are a large number of cells in the urine of the patient and all the cell sorters in the microfluidic chip are occupied, the spacing of 60 ± 3 μm allows excess cells to pass, thus avoiding the traditional filter method. The problem of confusion.
如图5所示，横向依次等边距排列的一个第一尺寸细胞分选器2-1、一个第二尺寸细胞分选器2-2和一个第三尺寸细胞分选器2-3组成一个第一细胞分选单元3-1；横向依次等边距排列的一个第一尺寸细胞分选器2-1、一个第三尺寸细胞分选器2-3和一个第二尺寸细胞分选器2-2组成一个第二细胞分选单元3-2；多个第一细胞分选单元3-1横向循环排布构成第一细胞分选阵列4-1，多个第二细胞分选单元3-2横向循环排布构成第二细胞分选阵列4-2，相互平行的一个第一细胞分选阵列和一个第二捕获阵列组成一个细胞分选二级阵列4，细胞捕获区域中多个相互平行的细胞分选二级阵列纵向等边距循环排布。As shown in FIG. 5, a first size cell sorter 2-1, a second size cell sorter 2-2 and a third size cell sorter 2-3 are arranged in a horizontally equilaterally spaced arrangement. First cell sorting unit 3-1; a first size cell sorter 2-1, a third size cell sorter 2-3, and a second size cell sorter 2 arranged in a laterally equilaterally spaced arrangement -2 constitutes a second cell sorting unit 3-2; a plurality of first cell sorting units 3-1 are arranged in a lateral circulation to constitute a first cell sorting array 4-1, and a plurality of second cell sorting units 3 - 2 The lateral circulation arrangement constitutes a second cell sorting array 4-2, a first cell sorting array and a second capturing array parallel to each other constitute a cell sorting secondary array 4, and a plurality of cells in the cell capturing region are parallel to each other The cell sorting secondary array is arranged in a longitudinal equilateral margin.
进一步地，在每个细胞分选二级阵列4中，第二细胞分选阵列4-2的第一尺寸细胞 分选器2-1的液流入口的中点与对应第一细胞分选阵列4-1的第三尺寸细胞分选器2-2和第二2-3尺寸细胞分选器横向边距的中点对齐。Further, in each of the cell sorting secondary arrays 4, the midpoint of the liquid inlet of the first size cell sorter 2-1 of the second cell sorting array 4-2 and the corresponding first cell sorting array The third size cell sorter 2-2 of 4-1 is aligned with the midpoint of the lateral margin of the second 2-3 size cell sorter.
进一步地，各奇数行细胞分选二级阵列的首行首列细胞分选器纵向位置对齐；各偶数行细胞分选二级阵列较相邻的前一奇数行细胞分选二级阵列向右侧等距偏移。有助于营造紊乱、随机的液流环境，避免细胞沿单一路径取向，从而提升微流控芯片的捕获效率。Further, the longitudinal rows of the first row of the first row of the odd-row cell sorting secondary arrays are aligned in the longitudinal direction; the even-numbered rows of the cells are sorted into the secondary arrays and the adjacent odd-numbered rows of cells are sorted into the second array to the right. Side isometric offset. Helps create a turbulent, random flow environment that avoids cell orientation along a single path, thereby increasing the capture efficiency of microfluidic chips.
流经本发明的前述微流控芯片的所有目标细胞如尿脱落细胞将以同等概率遭遇各级细胞分选器，从而达到“依据细胞尺寸差异分级捕获细胞”的目的。当细胞可以依据尺寸差异被分别捕获开来，则观察视野更整洁、细胞形态更清晰，有利于下一步针对细胞形态的分析判读(如现今临床通用的尿脱落细胞学检查)。All target cells such as urine exfoliated cells flowing through the aforementioned microfluidic chip of the present invention will encounter the cell sorters of the respective stages with equal probability, thereby achieving the purpose of "collecting cells according to the difference in cell size." When cells can be captured separately according to size differences, the observation field is more tidy and the cell morphology is clearer, which is conducive to the next step of analyzing the cell morphology (such as the current clinical urinary exfoliative cytology).
可按照聚二甲基硅氧烷(PDMS)标准工艺，采用常规载玻片作为基底，通过等离子处理键合，来制备本发明的微流控芯片。The microfluidic chip of the present invention can be prepared by a plasma treatment bond according to a standard process of polydimethylsiloxane (PDMS) using a conventional slide as a substrate.
PDMS是聚二甲基硅氧烷的英文缩写。聚二甲基硅氧烷属于固化型聚合物，固化型聚合物与固化剂混合后，经过一段时间可固化***得到一定结构的微流控芯片。PDMS is the abbreviation for polydimethylsiloxane. Polydimethylsiloxane is a curable polymer. After the curable polymer is mixed with a curing agent, it can be cured and hardened over a period of time to obtain a microfluidic chip with a certain structure.
先采用利用微流控芯片分选和捕获尿脱落细胞的优势在于，(1)经微流控芯片捕获的细胞依据尺寸差异而分布开来，使得观察视野清晰，方便病理医师阅片，从而规避了传统的滤膜法中所有细胞挤在一起而干扰后续阅片的风险；(2)染色时，所用试剂由微流注射泵控制，染色时间精确、染色过程自动化。The advantage of using microfluidic chip to sort and capture urine exfoliated cells is that (1) the cells captured by the microfluidic chip are distributed according to the size difference, so that the observation field is clear and convenient for the pathologist to read the film, thereby avoiding In the traditional membrane method, all the cells are squeezed together to interfere with the risk of subsequent reading; (2) When dyeing, the reagents used are controlled by a microfluidic injection pump, the dyeing time is accurate, and the dyeing process is automated.
综上所述，本发明的利用微流控芯片分选和捕获尿脱落细胞，并免疫荧光方法检测人尿液中的脱落肿瘤细胞，能够准确、客观地检测尿路上皮癌(包括膀胱癌、输尿管癌、肾盂癌)，且全程无创，并摆脱了对病理医生主观经验的依赖。因此，本发明有效克服了目前尿路上皮癌临床诊断中存在的敏感度低、依赖病理医生的主观判断、有创、低效等问题。In summary, the present invention utilizes a microfluidic chip to sort and capture urine exfoliated cells, and immunofluorescence method for detecting exfoliated tumor cells in human urine, which can accurately and objectively detect urothelial carcinoma (including bladder cancer, Ureteral cancer, renal pelvic cancer), and non-invasive throughout the process, and get rid of the dependence on the pathologist's subjective experience. Therefore, the invention effectively overcomes the problems of low sensitivity, subjective judgment, invasiveness and inefficiency of the pathologist according to the current clinical diagnosis of urothelial carcinoma.
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。Before the present invention is further described, it is to be understood that the scope of the present invention is not limited to the specific embodiments described below; It is not intended to limit the scope of the invention. The test methods which do not specify the specific conditions in the following examples are usually carried out according to conventional conditions or according to the conditions recommended by each manufacturer.
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。When the numerical values are given by the examples, it is to be understood that the two endpoints of each numerical range and any one of the two. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning meaning In addition to the specific methods, devices, and materials used in the embodiments, the methods, devices, and materials described in the embodiments of the present invention may also be used according to the prior art and the description of the present invention by those skilled in the art. Any method, apparatus, and material of the prior art, similar or equivalent, is used to practice the invention.
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring Harbor Laboratory Press，1989and Third edition，2001；Ausubel等，CURRENT PROTOCOLS IN MOLECULAR BIOLOGY，John Wiley&Sons，New York，1987and periodic updates；the series METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATIN STRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS IN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，Academic Press，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，Chromatin Protocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields conventional in the art. Conventional technology. These techniques are well described in the existing literature. For details, see Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons , New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman And AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, and the like.
实施例1微流控芯片捕获尿脱落细胞Example 1 Microfluidic chip captures exfoliated cells
如图1～5所示，本发明的微流控芯片，包括沿液流方向A依次连接的样本入口1-1、细胞捕获区域1-2以及样本出口1-3。细胞捕获区域1-2设有多个细胞分选器2，如图2所示，所述细胞分选器2由三个整体呈弧形排布的柱状凸起201构成，柱状凸起之间存在间隙，弧形开口202作为液流入口，中间柱状凸起两侧的间隙203作为液流出口，两个液流出口呈对称分布。细胞捕获区域1-2设有三种不同尺寸规格的细胞分选器2，分别为第一尺寸细胞分选器2-1、第二尺寸细胞分选器2-2和第三尺寸细胞分选器2-3，第一尺寸细胞分选器2-1、第二尺寸细胞分选器2-2和第三尺寸细胞分选器2-3的尺寸依次减小。其中，第一尺寸细胞分选器2-1的液流入口的宽度为60μm，两个液流出口的宽度均为22μm，可捕获直径在22～60μm范围内的细胞。第二尺寸细胞分选器2-1的液流入口的宽度为30μm，两个液流出口的宽度均为10μm，可捕获直径在10～22μm范围内的细胞。第三尺寸细胞分选器2-3的液流入口的宽度为16μm，两个液流出口的宽度均为4μm，可捕获直径在4～10μm范围内的细胞。相邻的细胞分选器尺寸不同，在整个细胞捕获区域1-2内营造紊乱的液流环境。As shown in FIGS. 1 to 5, the microfluidic chip of the present invention includes a sample inlet 1-1, a cell trapping region 1-2, and a sample outlet 1-3 which are sequentially connected in the liquid flow direction A. The cell trapping area 1-2 is provided with a plurality of cell sorters 2, as shown in Fig. 2, the cell sorter 2 is composed of three columnar protrusions 201 arranged in an overall arc shape, between the columnar protrusions There is a gap, the arcuate opening 202 serves as a liquid flow inlet, and the gap 203 on both sides of the middle columnar projection serves as a liquid flow outlet, and the two liquid flow outlets are symmetrically distributed. The cell capture zone 1-2 is provided with three different size cell sorters 2, a first size cell sorter 2-1, a second size cell sorter 2-2 and a third size cell sorter, respectively. 2-3, the sizes of the first size cell sorter 2-1, the second size cell sorter 2-2, and the third size cell sorter 2-3 are sequentially decreased. The liquid inlet of the first size cell sorter 2-1 has a width of 60 μm, and the widths of the two liquid flow outlets are both 22 μm, and cells having a diameter in the range of 22 to 60 μm can be captured. The flow inlet of the second-sized cell sorter 2-1 has a width of 30 μm, and both of the liquid outlets have a width of 10 μm, and cells having a diameter in the range of 10 to 22 μm can be captured. The flow inlet of the third-sized cell sorter 2-3 has a width of 16 μm, and both of the liquid outlets have a width of 4 μm, and cells having a diameter in the range of 4 to 10 μm can be captured. Adjacent cell sorters are sized differently to create a turbulent flow environment throughout the cell capture zone 1-2.
所述捕获区域1-2包括多个细胞分选单元3，每个所述细胞分选单元3由一个第一尺寸细胞分选器2-1、一个第二尺寸细胞分选器2-2和一个第三尺寸细胞分选器2-3组成。进一步地，位于同一行的细胞分选单元中各尺寸细胞分选器的排布方式相同。横向相邻的细胞分选器，边距d1相同，均为60μm。横向依次等边距排列的一个第一尺寸细胞分选器2-1、一个第二尺寸细胞分选器2-2和一个第三尺寸细胞分选器2-3组成一 个第一细胞分选单元3-1；横向依次等边距排列的一个第一尺寸细胞分选器2-1、一个第三尺寸细胞分选器2-3和一个第二尺寸细胞分选器2-2组成一个第二细胞分选单元3-2；多个第一细胞分选单元3-1横向循环排布构成第一细胞分选阵列4-1，多个第二细胞分选单元3-2横向循环排布构成第二细胞分选阵列4-2，相互平行的一个第一细胞分选阵列和一个第二捕获阵列组成一个细胞分选二级阵列4，细胞捕获区域中多个相互平行的细胞分选二级阵列纵向等边距(d3＝120μm)循环排布。The capture area 1-2 includes a plurality of cell sorting units 3, each of which is composed of a first size cell sorter 2-1, a second size cell sorter 2-2, and A third size cell sorter consists of 2-3. Further, the cell sorters of the respective sizes in the cell sorting unit located in the same row are arranged in the same manner. The laterally adjacent cell sorters have the same margin d1 and are all 60 μm. A first size cell sorter 2-1, a second size cell sorter 2-2 and a third size cell sorter 2-3 arranged in a horizontally equilaterally spaced arrangement form a first cell sorting unit 3-1; a first size cell sorter 2-1, a third size cell sorter 2-3, and a second size cell sorter 2-2 arranged side by side in a horizontally sequential manner The cell sorting unit 3-2; the plurality of first cell sorting units 3-1 are arranged in a horizontal circulation to constitute the first cell sorting array 4-1, and the plurality of second cell sorting units 3-2 are arranged in a horizontal circulation arrangement. The second cell sorting array 4-2, a first cell sorting array and a second capturing array parallel to each other constitute a cell sorting secondary array 4, and a plurality of mutually parallel cells in the cell capturing region are sorted into two The array is equilaterally spaced (d3 = 120 μm) in a circular arrangement.
各奇数行细胞分选二级阵列的首行首列细胞分选器纵向位置对齐；各偶数行细胞分选二级阵列较相邻的前一奇数行细胞分选二级阵列向右侧等距(d2＝50μm)偏移。按次规律，直至填满总长度和总宽度均为6000μm的微流控芯片捕获区域1-2。The first row of the first row of cell sorters in each odd row of cell sorting secondary arrays is aligned longitudinally; each even row of cell sorting secondary arrays is adjacent to the previous odd row of cells sorting secondary arrays to the right isometric (d2 = 50 μm) offset. The rule is repeated until the microfluidic chip capture area 1-2 having a total length and a total width of 6000 μm is filled.
按照聚二甲基硅氧烷(PDMS)标准工艺，采用常规2.5*7.5cm载玻片作为基底，通过等离子处理键合，来制备本发明的微流控芯片。The microfluidic chip of the present invention was prepared by a plasma treatment bonding using a conventional 2.5*7.5 cm glass slide as a substrate according to a standard procedure of polydimethylsiloxane (PDMS).
在注射泵的控制下，待处理的尿液样本或者尿沉渣的PBS(磷酸盐缓冲液)悬浮液，从样本入口1-1进入微流控芯片，途径细胞捕获区域1-2时尿脱落细胞被捕获，剩余液体从样本出口1-3流出微流控芯片。液体样本中的尿脱落细胞B随机流经各个不同尺寸规格的细胞分选器，不同尺寸的尿脱落细胞B被对应尺寸的细胞分选器2捕获。因此，不同尺寸的细胞在芯片内部规则分布开来，视野清晰，细胞形态完好，特别适用于下一步的分析。Under the control of the syringe pump, the urine sample to be treated or the PBS (phosphate buffer) suspension of urine sediment enters the microfluidic chip from the sample inlet 1-1, and the urine exfoliated cells pass through the cell capture zone 1-2. Captured, the remaining liquid exits the microfluidic chip from sample outlets 1-3. The urine exfoliated cells B in the liquid sample are randomly flowed through the cell sorters of different sizes, and the urine exfoliated cells B of different sizes are captured by the cell sorter 2 of the corresponding size. Therefore, cells of different sizes are regularly distributed inside the chip, the field of view is clear, and the cell morphology is intact, which is particularly suitable for the next analysis.
实施例2免疫荧光法判定尿脱落肿瘤细胞Example 2 Immunofluorescence assay for urinary exfoliated tumor cells
尿脱落细胞被实施例1中的微流控芯片捕获后，如图6所示，不同尺寸的细胞在芯片内部规则分布开来，视野清晰，细胞形态完好，特别适用于下一步的分析。After the urine exfoliated cells are captured by the microfluidic chip in Example 1, as shown in Fig. 6, cells of different sizes are regularly distributed inside the chip, the visual field is clear, and the cell morphology is intact, which is particularly suitable for the next analysis.
本实施例通过免疫荧光法来区别尿脱落肿瘤细胞与正常尿路上皮细胞。In this embodiment, urine detachment tumor cells and normal urothelial cells are distinguished by immunofluorescence.
免疫荧光染色步骤：多聚甲醛以0.5～4ml/h的流速通过捕获有尿脱落细胞的微流控芯片，维持30min；PBS以0.5～4ml/h的流速通过所述微流控芯片，维持3-5min；浓度为0.1％的Triton X-100溶液以0.5～4ml/h的流速通过所述微流控芯片，维持10min；PBS以0.5～4ml/h的流速通过所述微流控芯片，维持3-5min；BSA溶液以0.5～4ml/h的流速通过所述微流控芯片，维持30min。抗-CK20一抗(abcam,ab76126)、抗-CD44v6一抗(abcam,ab78960)混合溶液以0.5ml/h的流速通过所述微流控芯片，维持60min；PBS以0.5～4ml/h的流速通过微流控芯片，维持3-5min；CK20对应二抗(Invitrogen,A11070)、CD44v6对应二抗(abcam,ab150116)、DAPI(Invitrogen,MP01306)混合溶液以0.5ml/h的流速通过所述微流控芯片，维持30min；PBS以0.5～4ml/h的流速通过所述微流控芯片，维持3-5min。Immunofluorescence staining step: paraformaldehyde is passed through a microfluidic chip capturing urine exfoliated cells at a flow rate of 0.5-4 ml/h for 30 min; PBS is passed through the microfluidic chip at a flow rate of 0.5-4 ml/h to maintain 3 -5 min; a 0.1% Triton X-100 solution was passed through the microfluidic chip at a flow rate of 0.5 to 4 ml/h for 10 min; PBS was passed through the microfluidic chip at a flow rate of 0.5 to 4 ml/h. 3-5 min; BSA solution was passed through the microfluidic chip at a flow rate of 0.5 to 4 ml/h for 30 min. Anti-CK20 primary antibody (abcam, ab76126), anti-CD44v6 primary antibody (abcam, ab78960) mixed solution was passed through the microfluidic chip at a flow rate of 0.5 ml/h for 60 min; PBS was flowed at a flow rate of 0.5-4 ml/h. The microfluidic chip was maintained for 3-5 min; the CK20 corresponding secondary antibody (Invitrogen, A11070), CD44v6 corresponding secondary antibody (abcam, ab150116), DAPI (Invitrogen, MP01306) mixed solution passed through the micro at a flow rate of 0.5 ml/h. The flow control chip was maintained for 30 min; the PBS was passed through the microfluidic chip at a flow rate of 0.5 to 4 ml/h for 3-5 min.
最后，在荧光显微镜对应波长的激发光下，寻找CK20、CD44v6、DAPI三者同时阳性的细胞，为尿脱落肿瘤细胞。如图7所示。Finally, under the excitation light of the corresponding wavelength of the fluorescence microscope, the cells which were positive for CK20, CD44v6 and DAPI were found to be urinary exfoliated tumor cells. As shown in Figure 7.
具体地，CK20：激发波长450nm-480nm,发射波长515nm；CD44v6:激发波长515nm-585nm,发射波长610nm；DAPI:激发波长330nm-385nm,发射波长420nm。Specifically, CK20: excitation wavelength 450 nm to 480 nm, emission wavelength 515 nm; CD44v6: excitation wavelength 515 nm to 585 nm, emission wavelength 610 nm; DAPI: excitation wavelength 330 nm to 385 nm, emission wavelength 420 nm.
实施例3本发明的检测准确率Example 3 Detection accuracy of the present invention
为了验证本发明的准确率，我们收集了50位确诊的尿路上皮癌患者、13位非癌症志愿者的尿液采用上述实施例2中的方法进行检测。寻找CK20、CD44v6、DAPI三者同时阳性的细胞，为尿脱落肿瘤细胞。基于所有案例尿液中所检测到肿瘤细胞数目绘制ROC曲线(如图8所示)，得本发明的敏感度为：88.9％；特异度为：76.9％，ROC曲线下面积为0.808。To verify the accuracy of the present invention, we collected the urine of 50 confirmed urothelial carcinoma patients and 13 non-cancer volunteers using the method of Example 2 above. Look for cells that are positive at the same time as CK20, CD44v6, and DAPI, which are urinary exfoliated tumor cells. The ROC curve (shown in Figure 8) was plotted based on the number of tumor cells detected in urine in all cases. The sensitivity of the present invention was 88.9%; the specificity was 76.9%, and the area under the ROC curve was 0.808.
此外，我们也实施了与临床现役尿脱落细胞学检查的一对一比较。共采集17位尿路上皮癌确诊患者的尿液，其中一半尿液送往医院病理科进行尿脱落细胞学检查，另外一半尿液送往实验室采用本发明上述实施例2中的方法进行检测。寻找CK20、CD44v6、DAPI三者同时阳性的细胞，为尿脱落肿瘤细胞。结果为：17例确诊患者中，传统尿脱落细胞学检查呈阳性(包含疑似病例)的仅4例，准确率23.5％；而本发明在17位患者中检测出了15例阳性结果，准确率88.2％。详见下表1所示：In addition, we also performed a one-to-one comparison with clinical off-the-shelf urine exfoliative cytology. A total of 17 urine samples from patients diagnosed with urothelial carcinoma were collected. Half of the urine was sent to the hospital pathology department for urine exfoliative cytology. The other half was sent to the laboratory for testing according to the method of the above-mentioned Example 2 of the present invention. . Look for cells that are positive at the same time as CK20, CD44v6, and DAPI, which are urinary exfoliated tumor cells. The results were: in the 17 confirmed patients, only 4 cases of traditional urine exfoliative cytology were positive (including suspected cases), the accuracy rate was 23.5%; and the present invention detected 15 positive results in 17 patients, the accuracy rate 88.2%. See Table 1 below for details:
表1：本发明与传统尿脱落细胞学检查的比较Table 1: Comparison of the present invention with traditional urine exfoliative cytology
Figure PCTCN2018072880-appb-000004
Figure PCTCN2018072880-appb-000004
实施例4回收细胞进行单细胞测序Example 4 Recovering Cells for Single Cell Sequencing
对肿瘤细胞进行分子层面的分析具有巨大的临床价值，也充分体现了精准医疗的理念。 本发明的一大优势为：可无损捕获、回收单个尿脱落肿瘤细胞，从而为下游的单细胞测序打下基础。我们使用本发明上述实施例1中的微流控芯片捕获并回收了一位尿路上皮癌患者的6个尿脱落细胞(1个正常尿路上皮细胞、5个肿瘤细胞)，然后对该6个细胞分别进行单细胞测序，最后分析其拷贝数变异(CNV)情况。The molecular level analysis of tumor cells has great clinical value and fully reflects the concept of precision medicine. A major advantage of the present invention is that it can non-destructively capture and recover a single urinary exfoliated tumor cell, thereby laying the foundation for downstream single cell sequencing. We used the microfluidic chip of the above-mentioned Example 1 of the present invention to capture and recover 6 urine exfoliated cells (1 normal urothelial cell, 5 tumor cells) of a patient with urothelial carcinoma, and then 6 The cells were single-cell sequenced and finally analyzed for copy number variation (CNV).
如图9结果显示，肿瘤细胞均如预期出现了拷贝数紊乱的情况，而正常尿路上皮细胞的拷贝数则符合正常的二倍体特征。As shown in Fig. 9, the tumor cells showed a copy number disorder as expected, and the copy number of normal urothelial cells was consistent with the normal diploid characteristics.
这个例子充分证明了本发明可以高效地捕获并回收单个尿脱落细胞，全过程对目标细胞的损伤极小，可以衔接下游一系列的分子分析手段，因此具有重大价值与潜力。This example fully demonstrates that the present invention can efficiently capture and recover a single urinary exfoliated cell, and the damage to the target cell is minimal in the whole process, and can link a series of molecular analysis means downstream, thus having great value and potential.
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. It should be noted that those skilled in the art will also A number of improvements and additions may be made which are also considered to be within the scope of the invention. All of the equivalents of the changes, modifications, and evolutions that can be made by the above-disclosed technical content are all those skilled in the art without departing from the spirit and scope of the present invention. Equivalent embodiments; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above-described embodiments in accordance with the essential techniques of the present invention are still within the scope of the technical solutions of the present invention.

Claims (20)

  1. CK20蛋白和CD44v6蛋白共同用于制备或筛选尿脱落肿瘤细胞诊断试剂的用途。The use of CK20 protein and CD44v6 protein for the preparation or screening of diagnostic reagents for urine exfoliated tumor cells.
  2. 根据权利要求1所述的用途，其特征在于，CK20蛋白和CD44v6蛋白共同作为生物标志物。The use according to claim 1, characterized in that the CK20 protein and the CD44v6 protein are used together as a biomarker.
  3. 根据权利要求1所述的用途，其特征在于，CK20蛋白和CD44v6蛋白共同用于制备或筛选尿脱落肿瘤细胞诊断试剂的用途，包括两方面的内容，其一，CK20蛋白和CD44v6蛋白共同用于制备尿脱落肿瘤细胞诊断试剂，是指将CK20蛋白和CD44v6蛋白共同作为尿脱落肿瘤细胞的诊断指标应用于尿脱落肿瘤细胞诊断试剂的制备；CK20蛋白和CD44v6蛋白用于筛选尿脱落肿瘤细胞诊断试剂，是指将CK20蛋白和CD44v6蛋白共同作为尿脱落肿瘤细胞的识别靶标筛选分别或同时特异性识别CK20蛋白和CD44v6蛋白的试剂，从而作为尿脱落肿瘤细胞诊断试剂。The use according to claim 1, characterized in that the CK20 protein and the CD44v6 protein are used together for preparing or screening a diagnostic reagent for urine exfoliated tumor cells, including two aspects. First, the CK20 protein and the CD44v6 protein are used together. The preparation of diagnostic reagent for urine exfoliated tumor cells refers to the preparation of CK20 protein and CD44v6 protein as diagnostic indicators for urine exfoliated tumor cells in the preparation of diagnostic reagents for urine exfoliated tumor cells; CK20 protein and CD44v6 protein are used for screening urine cell tumor cell diagnostic reagents. The CK20 protein and the CD44v6 protein are used together as a recognition target for urinary exfoliated tumor cells to screen for the specific recognition of CK20 protein and CD44v6 protein, respectively, thereby serving as a diagnostic reagent for urinary exfoliated tumor cells.
  4. 根据权利要求1所述的用途，其特征在于，所述肿瘤为尿路上皮癌。The use according to claim 1, wherein the tumor is urothelial carcinoma.
  5. 分别或同时特异性识别CK20蛋白和CD44v6蛋白的试剂用于制备尿脱落肿瘤细胞诊断试剂盒的用途。The use of an agent for specifically recognizing CK20 protein and CD44v6 protein, respectively or simultaneously, for the preparation of a urine detachment tumor cell diagnostic kit.
  6. 根据权利要求5所述的用途，其特征在于，所述分别或同时特异性识别CK20蛋白和CD44v6蛋白的试剂选自分别或同时特异性结合CK20蛋白和CD44v6蛋白的抗体或配体。The use according to claim 5, characterized in that the agent which specifically or simultaneously recognizes the CK20 protein and the CD44v6 protein is selected from an antibody or a ligand which specifically or simultaneously binds specifically to the CK20 protein and the CD44v6 protein.
  7. 一种尿脱落肿瘤细胞诊断试剂盒，所述的试剂盒中至少包括分别或同时特异性识别CK20蛋白和CD44v6蛋白的试剂。A urine detachment tumor cell diagnostic kit comprising at least a reagent for specifically recognizing a CK20 protein and a CD44v6 protein, respectively or simultaneously.
  8. 根据权利要求7所述的试剂盒，其特征在于，所述分别或同时特异性识别CK20蛋白和CD44v6蛋白的试剂选自分别或同时特异性结合CK20蛋白和CD44v6蛋白的抗体或配体。The kit according to claim 7, wherein the agent that specifically or simultaneously recognizes the CK20 protein and the CD44v6 protein is selected from an antibody or a ligand that specifically binds to the CK20 protein and the CD44v6 protein, respectively or simultaneously.
  9. 根据权利要求7所述的试剂盒，其特征在于，所述试剂盒中包括：特异性结合CK20蛋白的第一抗体，特异性结合CD44v6的第一抗体，以及荧光素染料标记的分别特异性结合所述第一抗体的第二抗体。The kit according to claim 7, wherein said kit comprises: a first antibody that specifically binds to CK20 protein, a first antibody that specifically binds to CD44v6, and a specific binding of a fluorescein dye label, respectively a second antibody of the first antibody.
  10. 根据权利要求7所述的试剂盒，其特征在于，所述试剂盒中还包括细胞核荧光染料。The kit according to claim 7, wherein the kit further comprises a nuclear fluorescent dye.
  11. 根据权利要求7所述的试剂盒，其特征在于，所述试剂盒还包括微流控芯片，所述微流控芯片用于分选和捕获尿脱落细胞，所述微流控芯片包括依次连接的样本入口、细胞捕获区域以及样本出口，所述细胞捕获区域设有多个细胞分选器，所述细胞分选器 由三个整体呈弧形排布的柱状凸起构成，柱状凸起之间存在间隙，弧形开口作为液流入口，中间柱状凸起两侧的间隙作为液流出口，两个液流出口呈对称分布。The kit according to claim 7, wherein said kit further comprises a microfluidic chip for sorting and capturing urine exfoliated cells, said microfluidic chip comprising sequential connections a sample inlet, a cell capture region, and a sample outlet, the cell capture region being provided with a plurality of cell sorters, the cell sorter being composed of three columnar protrusions arranged in an overall arc shape, the columnar protrusions There is a gap between them, the arc-shaped opening serves as a liquid flow inlet, and the gap on both sides of the middle cylindrical protrusion serves as a liquid flow outlet, and the two liquid flow outlets are symmetrically distributed.
  12. 根据权利要求11所述的试剂盒，其特征在于，所述细胞捕获区域设有三种不同尺寸规格的细胞分选器，分别为第一尺寸细胞分选器、第二尺寸细胞分选器和第三尺寸细胞分选器，所述第一尺寸细胞分选器、第二尺寸细胞分选器和第三尺寸细胞分选器的尺寸依次减小。The kit according to claim 11, wherein said cell capture zone is provided with three different size cell sorters, a first size cell sorter, a second size cell sorter and a In the three-dimensional cell sorter, the size of the first size cell sorter, the second size cell sorter, and the third size cell sorter are sequentially decreased.
  13. 根据权利要求12所述的试剂盒，其特征在于，相邻的细胞分选器尺寸不同。The kit according to claim 12, wherein adjacent cell sorters are different in size.
  14. 根据权利要求13所述的试剂盒，其特征在于，所述微流控芯片包括多个细胞分选单元，每个所述细胞分选单元由一个第一尺寸细胞分选器、一个第二尺寸细胞分选器和一个第三尺寸细胞分选器组成。The kit according to claim 13, wherein said microfluidic chip comprises a plurality of cell sorting units, each of said cell sorting units being of a first size cell sorter, a second size The cell sorter is composed of a third size cell sorter.
  15. 根据权利要求14所述的试剂盒，其特征在于，位于同一行的细胞分选单元中各尺寸细胞分选器的排布方式相同。The kit according to claim 14, wherein the cell sorters of the respective sizes in the cell sorting unit in the same row are arranged in the same manner.
  16. 根据权利要求15所述的试剂盒，其特征在于，横向相邻的细胞分选器，边距相同。The kit according to claim 15, wherein the laterally adjacent cell sorters have the same margin.
  17. 根据权利要求16所述的试剂盒，其特征在于，横向依次等边距排列的一个第一尺寸细胞分选器、一个第二尺寸细胞分选器和一个第三尺寸细胞分选器组成一个第一细胞分选单元；横向依次等边距排列的一个第一尺寸细胞分选器、一个第三尺寸细胞分选器和一个第二尺寸细胞分选器组成一个第二细胞分选单元；多个第一细胞分选单元横向循环排布构成第一细胞分选阵列，多个第二细胞分选单元横向循环排布构成第二细胞分选阵列，相互平行的一个第一细胞分选阵列和一个第二捕获阵列组成一个细胞分选二级阵列，细胞捕获区域中多个相互平行的细胞分选二级阵列纵向等间距循环排布。The kit according to claim 16, wherein a first size cell sorter, a second size cell sorter and a third size cell sorter are arranged in a horizontally equilaterally spaced arrangement. a cell sorting unit; a first size cell sorter arranged in a laterally equilaterally spaced arrangement, a third size cell sorter and a second size cell sorter to form a second cell sorting unit; The first cell sorting unit is laterally arranged to form a first cell sorting array, and the plurality of second cell sorting units are laterally arranged to form a second cell sorting array, a first cell sorting array and one parallel to each other The second capture array constitutes a cell sorting secondary array, and a plurality of mutually parallel cell sorting secondary arrays in the cell capture region are arranged in a longitudinally equidistant circular arrangement.
  18. 根据权利要求17所述的试剂盒，其特征在于，在每个细胞分选二级阵列中，第二细胞分选阵列的第一尺寸细胞分选器的液流入口的中点与对应第一细胞分选阵列的第三尺寸细胞分选器和第二尺寸细胞分选器横向边距的中点对齐。The kit according to claim 17, wherein in each of the cell sorting secondary arrays, the midpoint of the liquid flow inlet of the first size cell sorter of the second cell sorting array and the corresponding first The third size cell sorter of the cell sorting array is aligned with the midpoint of the lateral margin of the second size cell sorter.
  19. 根据权利要求18所述的试剂盒，其特征在于，各奇数行细胞分选二级阵列的首行首列细胞分选器纵向位置对齐；各偶数行细胞分选二级阵列较相邻的前一奇数行细胞分选二级阵列向右侧等距偏移。The kit according to claim 18, wherein the first row of the first row of cell sorters of each of the odd row cell sorting secondary arrays is longitudinally aligned; each of the even rows of cells sorting the second array is adjacent to the front An odd-numbered row of cell sorting secondary arrays is offset equidistantly to the right.
  20. 根据权利要求12～19任一项所述的试剂盒，其特征在于，第一尺寸细胞分选器的液流入口的宽度为60±5μm，液流出口的宽度为22±5μm；第二尺寸细胞分选器的液流入口 的宽度为30±3μm，液流出口的宽度为10±3μm；第三尺寸细胞分选器的液流入口的宽度为16μ±1m，液流出口的宽度为4±1μm。The kit according to any one of claims 12 to 19, wherein the first inlet cell sorter has a liquid inlet having a width of 60 ± 5 μm and a liquid outlet outlet having a width of 22 ± 5 μm; The width of the flow inlet of the cell sorter is 30±3 μm, the width of the liquid outlet is 10±3 μm; the width of the liquid inlet of the third-sized cell sorter is 16 μ±1 m, and the width of the liquid outlet is 4 ±1 μm.
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