CN114047333A - Pancreatic cancer circulating tumor cell detection kit and application method thereof - Google Patents
Pancreatic cancer circulating tumor cell detection kit and application method thereof Download PDFInfo
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Abstract
The invention discloses a pancreatic cancer circulating tumor cell detection kit, which comprises: the kit comprises a micro-fluidic chip, a biotin-labeled antibody, an antibody diluent, a sample diluent, a cell fixing liquid, a cell permeation liquid and a cell fluorescent staining liquid, wherein a nano micro-array channel is arranged on a structural sheet of the micro-fluidic chip, and streptavidin magnetic particles are coated on the micro-array channel. The invention also discloses an application method of the pancreatic cancer circulating tumor cell detection kit. Compared with the prior art, the kit can be used for detecting pancreatic cancer circulating tumor cells, has higher sensitivity and specificity, can capture and separate the cells to keep integrity, can be used for subsequent cell analysis, and is convenient for early diagnosis, pathological typing, disease course staging and curative effect evaluation of pancreatic cancer.
Description
Technical Field
The invention belongs to the field of circulating tumor cell detection, and particularly relates to a pancreatic cancer circulating tumor cell detection kit and an application method thereof.
Background
Tumors are a serious health-threatening group of diseases with high morbidity and mortality. Pancreatic cancer is a digestive system tumor with extremely high malignancy, and the prognosis of patients is extremely poor due to no obvious symptoms in early stage, fast disease progression and low chemotherapy efficiency.
Circulating Tumor Cells (CTCs) can be used for developing and analyzing various tumor cells, and micro lesions can be found in the image diagnosis, which is an important basis for the judgment of the progress and infiltration and poor prognosis of various tumor diseases. The circulating tumor cells have important values in the aspects of tumor early diagnosis, individualized treatment/curative effect evaluation, tumor metastasis mechanism research and the like. However, because the proportion of the circulating tumor cells in the human body is very low, the separation and identification of the circulating tumor cells become a difficult point in clinical application. The heterogeneity of circulating tumor cells limits the applicability of traditional methods that rely on epithelial-specific antigen expression to identify CTCs, and can only detect EpCAM-positive cells, not EpCAM-negative cells. Flow Cytometry (FCM) is a method for analyzing cells by a Flow cytometer based on the principle that a fluorescent-labeled monoclonal antibody binds to a specific antigen on the surface of a cell. The flow cytometry method has fast detection speed, can realize the quantitative detection of tumor cells, and can perform multi-parameter analysis on the same cell.
The microfluidic chip is a CTCs separation method with advanced technology, can independently operate and process cells, accurately captures rare cells in a mixture, and can efficiently separate live cancer cells from blood. The microfluidic chip pipeline can be designed according to the size of cells, and can also separate CTCs by utilizing the specific binding of immunomagnetic beads and antibodies, and the CTCs captured by the binding of antigen and antibody can be released by trypsin. The microfluidic chip has the advantages of high sensitivity, low cost, miniature equipment and the like, the cell morphology is not damaged, and the separated CTCs can be used for subsequent in vitro culture and cell analysis.
In the prior art, a kit capable of detecting pancreatic cancer circulating tumor cells is lacked, which is not beneficial to early diagnosis, pathological typing, disease course staging and curative effect evaluation of pancreatic cancer.
Disclosure of Invention
The invention aims to: the pancreatic cancer circulating tumor cell detection kit and the application method thereof can be used for detecting pancreatic cancer circulating tumor cells, have higher sensitivity and specificity, capture and separate cells to keep integrity, can be used for subsequent cell analysis, and are convenient for early diagnosis, pathological typing, disease course staging and curative effect evaluation of pancreatic cancer.
In order to achieve the above objects, in one aspect, the present invention provides a pancreatic cancer circulating tumor cell detection kit, comprising: the kit comprises a micro-fluidic chip, a biotin-labeled antibody, an antibody diluent, a sample diluent, a cell fixing liquid, a cell permeation liquid and a cell fluorescent staining liquid, wherein a nano micro-array channel is arranged on a structural sheet of the micro-fluidic chip, and streptavidin magnetic particles are coated on the micro-array channel.
As a further description of the above technical solution:
the components of the biotin-labeled antibody include a biotin-labeled mouse anti-HLA-G antibody, a biotin-labeled mouse anti-CA 19-9 antibody, and a biotin-labeled mouse anti-CA 125 antibody.
As a further description of the above technical solution:
the components of the antibody diluent comprise Tris-HCL buffer solution, bovine serum albumin and sodium azide.
As a further description of the above technical solution:
the sample diluent is phosphate buffer.
As a further description of the above technical solution:
the cell fixing solution is 4% paraformaldehyde solution.
As a further description of the above technical solution:
the cells were permeabilized with 0.2% Triton-X100.
As a further description of the above technical solution:
the components of the cell fluorescent staining solution comprise a cytokeratin antibody containing a fluorescent labeling antibody Alexa Fluor488 fluorescein label, a PE labeled anti-human CD45 antibody and a nucleic acid dye DAPI.
On the other hand, the invention also provides an application method of the pancreatic cancer circulating tumor cell detection kit, which comprises the following steps:
s1 separation of peripheral blood mononuclear cells
a) Taking 10ml of sample, putting into a 50ml test tube, and gently mixing;
b) standing the test tube in an incubator at room temperature or 37 deg.C until the red blood cells naturally settle;
c) sucking the cell suspension rich in the white blood cells on the red blood cell layer by using a capillary tube, and transferring the cell suspension into another test tube;
d) adding Ca-free 2+ Mg2+ Hank's solution to a position 3cm away from the test tube port, uniformly mixing, centrifuging, removing supernatant, and repeatedly washing twice by the same method;
e) the precipitated cells are re-suspended by using a proper amount of 10-20% Hank's solution of inactivated calf serum, and the cell suspension is used as a processed sample for subsequent detection;
s2 coating of capture antibodies
a) Cleaning the microfluidic chip by using a sample diluent;
b) adding a biotin-labeled antibody into a PBS sterile buffer solution, uniformly mixing to obtain an antibody capture working solution, injecting the antibody capture working solution into a microfluidic chip by using an injector, and then placing the microfluidic chip in a wet box for incubation;
c) washing the micro-fluidic chip coated with the capture antibody by using a phosphate sterile buffer solution to remove the capture antibody working solution remained in the micro-fluidic chip;
d) sealing the chip;
s3 Capture of circulating tumor cells
a) Washing the microfluidic chip with sterile sample diluent for 3 times;
b) injecting the cell suspension prepared in the step S1 into the coated microfluidic chip by using an injector, and standing at room temperature;
c) injecting the cell fixing solution into the microfluidic chip by using an injector, and standing;
d) washing the microfluidic chip with a sterile sample diluent;
s4 immunofluorescence staining of circulating tumor cells
a) Washing the microfluidic chip with a sterile sample diluent;
b) injecting the cell fluorescent staining solution containing the antibody marker into the microfluidic chip by using an injector, and incubating;
c) washing the microfluidic chip with a sterile sample diluent;
d) injecting a nucleic acid fluorescent coloring agent into the microfluidic chip, and standing;
e) and (5) observing under a fluorescence inverted microscope.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. in the invention, the microarray channel of the microfluidic chip in the kit is coated with streptavidin magnetic particles, and streptavidin is a tetramer protein which can be connected with more biotinylated molecules to form a complex similar to crystal lattices, so that reaction signals are amplified, and the sensitivity of circulating tumor cell detection is greatly improved.
2. In the kit, the antibody diluent consists of Tris-HCL buffer solution, bovine serum albumin and sodium azide. Bovine serum albumin can maintain the activity and stability of the antibody, reduce non-specific binding, reduce non-specific background coloring and improve the specificity and sensitivity of the circulating tumor cell detection experiment.
3. In the invention, the microfluidic chip is matched with an antibody capture working solution prepared from a biotin-labeled mouse anti-HLA-G antibody, a biotin-labeled mouse anti-CA 19-9 antibody, a biotin-labeled mouse anti-CA 125 antibody and a sterile buffer solution of 1 XPBS (phosphate buffer solution), so that the captured and separated cells keep the integrity, and the microfluidic chip can be used for subsequent cell analysis and is convenient for early diagnosis of pancreatic cancer, pathological typing, staging of disease course and evaluation of curative effect.
Drawings
Fig. 1 is a graph of fluorescence staining of CTC cells at 400 x magnification under a microscope.
A: DAPI-labeled nuclei appeared blue; b: CK18 labeled tumor cells showed green shade; c: CD45 labeled leukocytes, appearing in red; d: when DlPA (+)/CK18(+)/CD45(-) appeared, the cells were tumor cells.
Detailed Description
Exemplary embodiments of the present disclosure will be described in more detail below. While exemplary embodiments of the present disclosure have been shown, it should be understood that the present disclosure may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
In one aspect, the invention provides a pancreatic cancer circulating tumor cell detection kit, comprising: the kit comprises a micro-fluidic chip, a biotin-labeled antibody, an antibody diluent, a sample diluent, a cell fixing liquid, a cell permeation liquid and a cell fluorescent staining liquid, wherein a nano micro-array channel is arranged on a structural sheet of the micro-fluidic chip, and streptavidin magnetic particles are coated on the micro-array channel.
The biotin-avidin system is a signal amplification labeling technology with the advantages of high affinity, high sensitivity, strong specificity, good stability and the like. Because the microarray channel of the microfluidic chip is coated with streptavidin magnetic particles, streptavidin is a protein secreted by streptomyces, is a tetramer protein, can be closely combined with 4 biotin molecules, and can be connected with more biotinylated molecules to form a complex similar to a crystal lattice, so that a reaction signal is amplified, and the detection sensitivity is greatly improved.
The biotin-labeled antibody contains a biotin-labeled mouse anti-HLA-G antibody, a biotin-labeled mouse anti-CA 19-9 antibody and a biotin-labeled mouse anti-CA 125 antibody. Human leukocyte antigen G (HLA-G) is an atypical MHC class I molecule having seven protein isoforms. HLA-G is used as an immune tolerance molecule and can act with a plurality of immunocompetent cells in vivo, thereby achieving the effect of weakening immune response. Under physiological conditions, HLA-G is expressed only in precursors of placental trophoblasts, thymus, cornea, nail stroma, pancreas, erythrocytes, and endothelial cells. In pathological conditions, HLA-G can be abnormally expressed on tumors, viral infections, autoimmune diseases and monocytes. HLA-G is expressed in most pancreatic cancer patients, and sHLA-G can be used as an independent prognostic factor and is a valuable prognostic indicator. CA19-9 is a kind of constitutive expression glucoprotein, and can be expressed in the serum of normal human and cancer patient with cancer of pancreatic cancer, gastric cancer, etc. But the expression level in normal human serum is generally low. CA19-9 has high relativity with pancreatic cancer, can reach about 70%, is a serological tumor marker which is most widely applied to pancreatic cancer, and is conventionally used for assisting in diagnosing pancreatic cancer or evaluating the treatment effect of pancreatic cancer. CA125 is a tumor-associated antigen that has been demonstrated by monoclonal techniques, is a glycoprotein in nature, and is present in tissues derived from the epithelium of body cavities such as the peritoneum, pleura, and pericardium. CA125 is the first carbohydrate antigen tumor marker for pancreatic, endometrial and ovarian cancer. When other cancers such as intestinal cancer occur, the serum content of the cancer usually rises.
Antibody dilution: consists of Tris-HCL buffer solution, bovine serum albumin and sodium azide. Bovine serum albumin can maintain the activity and stability of the antibody, reduce non-specific binding, reduce non-specific background coloring and improve the specificity and sensitivity of the experiment.
The sample diluent is phosphate buffer.
The cell fixing solution is 4% paraformaldehyde solution.
The cell permeation solution was 0.2% Triton-X100.
Cell fluorescent staining solution: contains a fluorescence labeling antibody Alexa Fluor488 fluorescein labeled cytokeratin antibody (Alexa Fluor488-anti-CK18), a PE labeled anti-human CD45 antibody (PE-anti-CD45) and a nucleic acid dye DAPI.
On the other hand, the invention also provides an application method of the pancreatic cancer circulating tumor cell detection kit, which comprises the following specific detection steps:
s1 separation of peripheral blood mononuclear cells
1) Taking 10ml of peripheral blood into a centrifugal tube with 50m of anticoagulant, and gently mixing the peripheral blood and the centrifugal tube;
2) standing the test tube vertically in an incubator at room temperature or 37 ℃ for 30-60 min until the red blood cells naturally settle;
3) sucking the cell suspension rich in the white blood cells on the red blood cell layer by using a capillary tube, and transferring the cell suspension into another test tube;
4) adding Ca-free 2+ Mg2+ Hank's solution to a position 3cm away from the test tube port, uniformly mixing, centrifuging for 10min at 2000r/min by using a horizontal centrifuge, removing supernatant, and washing twice by using the same method;
5) the precipitated cells are re-suspended by using a proper amount of 10-20% Hank's solution of inactivated calf serum to prepare cell suspension which is used as a processed sample for subsequent detection;
s2 coating of capture antibodies
1) Washing the microfluidic chip with 200 μ L phosphate buffer (taking out the chip in advance and balancing to room temperature);
2) the biotin-labeled mouse anti-HLA-G antibody, the biotin-labeled mouse anti-CA 19-9 antibody and the biotin-labeled mouse anti-CA 125 antibody were added to a1 XPBS sterile buffer solution, respectively, to prepare an antibody capture working solution with a concentration of 5. mu.g/ml. Respectively taking 100 mu l of the mixture and uniformly mixing the mixture to obtain mixed antibody capture working solution;
injecting 100 mu L of mixed antibody capture working solution into the chip by using an injector, then placing the chip into a wet box, and incubating overnight at 4 ℃; in the whole incubation process, all chip flow channels are ensured to be covered with antibody capture working solution all the time;
3) washing the chip coated with the capture antibody with 200. mu.L of 1 XPBS sterile buffer solution for 3 times, and removing the working solution of the capture antibody remaining in the chip;
4) blocking the chip by using 5% BSA solution at 27 ℃ for 2 hours, and finishing the coating of the capture antibody on the chip;
s3 Capture of circulating tumor cells
1) Washing the chip with sterile phosphate buffer solution for 3 times;
2) injecting the prepared cell suspension into the coated chip by using an injector, and standing for 30min at room temperature;
3) washing the chip with sterile phosphate buffer solution for 3 times;
s4 immunofluorescence staining of circulating tumor cells
1) The chip was washed 3 times with 200. mu.l of 1 XPBS sterile phosphate buffer;
2) injecting 200 μ l of 4% paraformaldehyde solution into the chip by using a syringe, and standing for 2 hours to fix the cells captured on the chip;
3) the chip was washed 3 times with 200. mu.l of 1 XPBS sterile phosphate buffer;
4) injecting 200 μ l of 0.2% Triton-X100 into the chip with a syringe to permeabilize the cells;
5) injecting 200 μ l of Alexa Fluor488 fluorescein-labeled cytokeratin antibody (Alexa Fluor488-anti-CK18) and PE-labeled anti-human CD45 antibody (PE-anti-CD45) labeled cytofluorescent staining solution into the chip by using a syringe, and incubating for 2 hours;
7) the chip was washed 3 times with 200. mu.l of 1 XPBS sterile phosphate buffer;
8) injecting 200 μ l DAPI fluorescent staining agent into the chip with a syringe, standing for 15 min;
9) the image was observed under a fluorescent inverted microscope, and a fluorescent stain image (see FIG. 1) was recorded and analyzed.
Results
Three-color immunofluorescence staining was performed with DAPI, Alexa Fluor 488-labeled anti-Cytokeratin (CK), and Alexa Fluor 555-labeled anti-CD45, and positive standards for CTCs were determined to be DAPI-positive, CK-positive, and CD 45-negative. While the positive criteria for leukocytes were DAPI positive, CK negative and CD45 positive. The experimental results show that: the staining result of the detected tumor cells is positive, and the tumor cells can be detected.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (8)
1. A pancreatic cancer circulating tumor cell detection kit, characterized by comprising: the kit comprises a micro-fluidic chip, a biotin-labeled antibody, an antibody diluent, a sample diluent, a cell fixing solution, a cell permeation solution and a cell fluorescent staining solution, wherein a nano micro-array channel is arranged on a structural sheet of the micro-fluidic chip, and streptavidin magnetic particles are coated on the micro-array channel.
2. The kit of claim 1, wherein the components of the biotin-labeled antibody include a biotin-labeled mouse anti-HLA-G antibody, a biotin-labeled mouse anti-CA 19-9 antibody, and a biotin-labeled mouse anti-CA 125 antibody.
3. The kit for detecting circulating tumor cells of pancreatic cancer according to claim 1, wherein the components of the antibody diluent comprise Tris-HCl buffer, bovine serum albumin and sodium azide.
4. The kit for detecting circulating tumor cells of pancreatic cancer according to claim 1, wherein the sample diluent is phosphate buffered saline.
5. The kit of claim 1, wherein the cell fixative is 4% paraformaldehyde solution.
6. The kit of claim 1, wherein the cell permeability is 0.2% Triton-X100.
7. The kit for detecting pancreatic cancer circulating tumor cells as claimed in claim 1, wherein the components of said cytofluorescent staining solution comprise cytokeratin antibody labeled with fluorescence labeling antibody Alexa Fluor488 fluorescein, PE labeled anti-human CD45 antibody and nucleic acid dye DAPI.
8. An application method of a pancreatic cancer circulating tumor cell detection kit is characterized by comprising the following steps:
s1 separation of peripheral blood mononuclear cells
a) Taking 10ml of sample, putting into a 50ml test tube, and gently mixing;
b) standing the test tube in an incubator at room temperature or 37 deg.C until the red blood cells naturally settle;
c) sucking the cell suspension rich in the white blood cells on the red blood cell layer by using a capillary tube, and transferring the cell suspension into another test tube;
d) adding Ca-free 2+ Mg2+ Hank's solution to a position 3cm away from the test tube port, uniformly mixing, centrifuging, removing supernatant, and repeatedly washing twice by the same method;
e) the precipitated cells are re-suspended by using a proper amount of 10-20% Hank's solution of inactivated calf serum, and the cell suspension is used as a processed sample for subsequent detection;
s2 coating of capture antibodies
a) Cleaning the microfluidic chip by using a sample diluent;
b) adding a biotin-labeled antibody into a PBS sterile buffer solution, uniformly mixing to obtain an antibody capture working solution, injecting the antibody capture working solution into a microfluidic chip by using an injector, and then placing the microfluidic chip in a wet box for incubation;
c) washing the micro-fluidic chip coated with the capture antibody by using a phosphate sterile buffer solution to remove the capture antibody working solution remained in the micro-fluidic chip;
d) sealing the chip;
s3 Capture of circulating tumor cells
a) Washing the microfluidic chip with sterile sample diluent for 3 times;
b) injecting the cell suspension prepared in the step S1 into the coated microfluidic chip by using an injector, and standing at room temperature;
c) injecting the cell fixing solution into the microfluidic chip by using an injector, and standing;
d) washing the microfluidic chip with a sterile sample diluent;
s4 immunofluorescence staining of circulating tumor cells
a) Washing the microfluidic chip with a sterile sample diluent;
b) injecting the cell fluorescent staining solution containing the antibody marker into the microfluidic chip by using an injector, and incubating;
c) washing the microfluidic chip with a sterile sample diluent;
d) injecting a nucleic acid fluorescent coloring agent into the microfluidic chip, and standing;
e) and (5) observing under a fluorescence inverted microscope.
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