CN111518767A

CN111518767A - Method for specific separation, identification and detection of CTC in biliary tract tumor serum

Info

Publication number: CN111518767A
Application number: CN202010162771.6A
Authority: CN
Inventors: 易滨; 梁晓飞; 武田�; 汪新燕; 刘林春
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-03-10
Filing date: 2020-03-10
Publication date: 2020-08-11

Abstract

The invention provides a method for specifically sorting CTC in biliary tract tumor serum, which comprises the steps of taking a proper amount of peripheral blood from a biliary tract tumor patient, placing the peripheral blood in an anticoagulation centrifugal tube, and uniformly mixing a whole blood sample; sequentially carrying out sample treatment of removing plasma protein and plasma nucleic acid, removing red blood cells, layering and centrifuging and removing white blood cells on the collected blood sample; the use of an antibody of the HSPG or SDC1 subtype or an antibody of the SDC2 subtype or an antibody of the GPC1 subtype or an antibody of the GPC3 subtype in combination with immunomagnetic beads to capture CTCs that are HSPG positive or SDC1 positive or SDC2 positive or GPC1 positive or GPC3 positive. The invention takes HSPG as a functional tumor molecular marker for sorting the relatively specific CTC of the gallbladder cancer and the bile duct cancer, thereby improving the sorting efficiency of the CTC of the biliary tract tumor, establishes a set of CTC molecular typing scoring system based on the HSPG, obtains a detection method for high specificity and high sensitivity of the biliary tract tumor, carries out downstream RNA-seq and gene NGS detection on the basis of the efficiently sorted CTC of the biliary tract tumor, and obtains tumor specific molecular information for improving the specificity.

Description

Method for specific separation, identification and detection of CTC in biliary tract tumor serum

Technical Field

The invention relates to the technical field of biological and medical detection, in particular to a method for specifically sorting, identifying and detecting CTC in biliary tract tumor serum.

Background

Circulating Tumor Cells (CTCs) in human peripheral blood refer to Tumor cells that spread from a Tumor lesion into the peripheral blood circulation and can develop into a metastatic lesion of the Tumor under certain conditions. Since more than 90% of cancer deaths are caused by metastasis, CTCs are a direct source of tumor metastasis, the isolation and molecular detection of CTCs from blood is becoming increasingly important.

The detection of CTCs has been expanded from simple cell counting to various directions of downstream cluster analysis, molecular typing, gene, transcription and protein level detection, and has entered clinical practice in breast cancer, colorectal cancer, prostate cancer and lung cancer, but there is still a great deal of blank in the diagnosis and treatment of biliary tract cancer. In the precise medical age, the blood marker detection has the following characteristics and advantages, and provides a new idea for a tumor-related gene mutation detection method, 1. the sampling is simple, and the follow-up rate can be improved; 2. the detection and follow-up observation can be carried out for multiple times, and the detection rate of malignant tumors can be increased; 3. the medicine has no radioactive and traumatic damages, and can reduce the incidence rate of complications; 4. can be used for monitoring relapse drug resistance after treatment, and improving treatment efficiency. 5. The inspection cost is reduced, the economic pressure of governments and patients is relieved, and the economic benefit ratio is high.

Heparan Sulfate Proteoglycan (HSPG) is a group of glycoproteins bound to core proteins by Heparan sulfate chains, distributed on the cell surface and in the extracellular matrix, the matrix HSPG being mainly associated with cellular infiltration and migration; the cytomembrane HSPG mainly comprises syndecano and Glypican, and the functions of the cytomembrane HSPG relate to: co-receptors as growth factors regulate signal transduction, co-regulate cell migration with adhesion molecules, bind to regulate extracellular matrix metalloproteinase activity, and the like. In tumor development and progression, the content, composition and function of HSPG are abnormal, and membrane HSPG can bind with various growth factors (such as FGF, PDGF, EGF, HGF, IGF and the like) and activate related channels. Overall HSPG expression has been rarely studied in biliary tract cancer, and we have obtained results including: 527 gallbladder cancer tissue specimens show that the total HSPG is mainly expressed on the surface of gallbladder cancer cells, and the positive rate of the total HSPG expression is 77.0 percent (406/527). In the low-differentiation adenocarcinoma, the HSPG content is increased, the cell differentiation degree is low, local metastasis is accompanied, the HSPG sulfation level in GBC tissues at the late stage of clinical staging is increased, and the sulfation HSPG positive patients have poorer chemotherapy reactivity and shorter survival time.

Compared with tissues, circulating tumor cells can realize non-invasive detection, and compared with free macromolecules in blood plasma, the circulating tumor cells have incomparable stability, so that the circulating tumor cells have great advantages and potentials. The invention takes HSPG as a functional tumor molecular marker for sorting the relatively specific CTC of the gallbladder cancer and the bile duct cancer, thereby improving the sorting efficiency of the CTC of the biliary tract tumor, establishes a set of CTC molecular typing scoring system based on the HSPG, obtains a detection method for high specificity and high sensitivity of the biliary tract tumor, carries out downstream RNA-seq and gene NGS detection on the basis of the efficiently sorted CTC of the biliary tract tumor, and obtains tumor specific molecular information for improving the specificity.

Disclosure of Invention

The invention aims to provide a method for specific sorting, identifying and detecting CTC in biliary tract tumor serum, which takes HSPG as a functional tumor molecular marker for sorting relatively specific CTC of biliary tract cancer and cholangiocarcinoma so as to improve the sorting efficiency of the biliary tract tumor CTC, establishes a set of CTC molecular typing and scoring system based on the HSPG, obtains a detection method for high specificity and high sensitivity of biliary tract tumor, detects downstream RNA-seq and gene NGS based on the efficiently sorted biliary tract tumor CTC, and obtains tumor specific molecular information for improving specificity.

The technical scheme adopted by the invention is as follows: a method for specifically sorting CTC in serum of biliary tract tumor comprises the following steps:

(1) sampling: taking a proper amount of peripheral blood from a biliary tract tumor patient, placing the peripheral blood into an anticoagulation centrifugal tube, and uniformly mixing a whole blood sample;

(2) sequentially carrying out sample treatment of removing plasma protein and plasma nucleic acid, removing red blood cells, carrying out layered centrifugation and removing white blood cells on the blood sample collected in the step (1);

(3) washing the magnetic particles: sucking a proper amount of immune magnetic particle suspension into an EP (EP) tube, standing, sucking the solution, adding a buffer solution, mixing uniformly, standing, removing a supernatant, repeating for 3 times, and then resuspending the magnetic particles to the original volume by using the buffer solution;

(4) antibody incubation: slowly adding a certain amount of immune magnetic particles into the sample treated in the step (2), reversing and uniformly mixing, adjusting the shaking speed of a shaking table, obliquely fixing a centrifugal tube on the shaking table, shaking at room temperature for 30min, and uniformly mixing every 10 min;

(5) enriching CTC: transferring the processed sample in the step (4) to a new centrifugal tube, standing the centrifugal tube on a magnetic frame, adsorbing magnetic beads for capturing CTC on one side of the magnetic frame, inverting the magnetic frame and the centrifugal tube, and cleaning a sample on a centrifugal tube cover;

(6) after standing for 10min, the clear solution was aspirated off, followed by the addition of an appropriate amount of dd H₂O cleaning the magnetic ball;

(7) adding a CF1 fixing solution for smear, and drying at 37 ℃;

(8) dropwise adding 4% paraformaldehyde for fixation for 8min, and preheating in a water bath at 37 deg.C containing 2 XSSC buffer solution for 10 min;

(9) respectively dehydrating with 75%, 85% and anhydrous ethanol for 2min, and air drying at room temperature;

(10) adding 10 μ L of fluorescent probe and mounting, hybridizing at 76 deg.C for 10min, and hybridizing at 37 deg.C for 1.5 h;

(11) tearing off the sealing glue by using a forceps, putting the sealing glue into formamide at the temperature of 43 ℃, peeling off a cover glass, soaking and washing for 10min, then soaking and washing for 15min by using 2 XSSC buffer solution, and shaking once every 5 min;

(12) coating 100 mu L of CK fluorescent antibody staining solution on the sample area, and incubating for 1h under the condition of keeping away from light in a wet box at 37 ℃;

(13) the CK fluorescent antibody staining solution was washed away, washed twice with 0.2% BSA, and then 10. mu.L of LDAPI coverslip was added.

Preferably, the processes of removing plasma proteins and plasma nucleic acids, removing red blood cells, performing stratified centrifugation and removing white blood cells in the blood sample processing in the step (2) are specifically as follows:

s1: removing plasma protein and plasma nucleic acid, adding the collected blood sample into buffer solution, centrifuging to remove supernatant, then gently shaking the centrifuge tube, and uniformly mixing precipitated cells;

s2: removing red blood cells: uniformly mixing the precipitated cells, adding a lysis solution, uniformly mixing the anticoagulation centrifugal tube in a vertical mixing instrument, centrifuging to remove supernatant, slightly shaking the anticoagulation centrifugal tube, uniformly mixing the precipitated cells, and adding a buffer solution;

s3: layering and centrifuging: adding a layering liquid into a new anticoagulation centrifugal tube, superposing substances in the anticoagulation centrifugal tube in S2 on the upper layer of the layering liquid, cleaning the tube wall of the anticoagulation centrifugal tube in S2 by using a buffer solution, simultaneously transferring the cleaning solution to the top layer of the layering liquid, and then performing centrifugal treatment;

s4: and (3) removing white blood cells: and (3) centrifuging the anticoagulation centrifuge tube in S3 to obtain three layers of solution, gently sucking the uppermost 2 layers of solution into a new centrifuge tube, adding a buffer solution, reversing and uniformly mixing, centrifuging to remove supernatant, then adding the buffer solution, and uniformly mixing and precipitating cells.

Preferably, the immunomagnetic particles in step (3) are one of antibodies bound to HSPG, antibodies of the SDC1 subtype, antibodies of the SDC2 subtype, antibodies of the GPC1 subtype and antibodies of the GPC3 subtype.

Preferably, the immunomagnetic particles to which the HSPG antibodies are bound consist of: iron oxide nanoparticles, cholestrol, GHDC, DOPC, HSPG.

Preferably, the immunomagnetic particles bound with the SDC1 subtype antibody consist of the following components: iron oxide nanoparticles, cholestrol, GHDC, DOPC, SDC 1.

Preferably, the immunomagnetic particles bound with the SDC2 subtype antibody consist of the following components: iron oxide nanoparticles, cholestrol, GHDC, DOPC, SDC 2.

Preferably, the immunomagnetic particles combined with the GPC1 subtype antibody consist of the following components: iron oxide nanoparticles, cholestrol, GHDC, DOPC, GPC 1.

Preferably, the immunomagnetic particles combined with the GPC3 subtype antibody consist of the following components: iron oxide nanoparticles, cholestrol, GHDC, DOPC, GPC 3.

Preferably, 10 μ L of immunomagnetic particles are taken in step (4) and mixed in 7.5mL of sample by inversion.

A method for identifying and detecting CTC in biliary tract tumor serum adopts QIAGEN kit to extract DNA based on circulating tumor cells, and comprises the following steps:

(a) taking 10ml of the blood sample treated in the step (2), adding 2.5 times of Solution A, reversing, uniformly mixing, centrifuging at 8000rpm for 5 minutes, removing supernatant, and repeating the steps for 1-2 times;

(b) preparing a mixed Solution of Solution B and Solution C according to information provided by an attached table of a QIAGEN kit;

(c) adding 5ml of the mixed Solution of Solution B and Solution C, blowing and beating uniformly by using a disposable plastic straw, incubating in a water bath or an incubator at 60 ℃ for 10 minutes, and turning over and mixing uniformly once during incubation, wherein the Solution is changed from red to yellow green or brown;

(d) adding isopropanol with the same volume, fully reversing and uniformly mixing until filamentous or flocculent DNA appears;

(e) transferring the floccule into a 1.5ml EP tube added with 800 mu L of 75% alcohol by using a pipette, inverting for several times, centrifuging for 1 minute at 12000prm, discarding the supernatant, and drying for 10-15 minutes at room temperature;

(f) adding 1000 mu L of Elution buffer, incubating for 10-15 minutes at 60 ℃ or dissolving DNA overnight, blowing and beating uniformly by using a pipette or a disposable plastic pipette, and storing the DNA at-20 ℃;

(g) and (3) carrying out DNA mutation detection of the biliary tract tumor blood circulation tumor cell source by adopting a high-throughput sequencing method.

The invention has the advantages that: the invention adopts a positive capture method of combining antigen and antibody, and carries out separation and enrichment of CTC through immunomagnetic beads. Capturing HSPG-positive or SDC 1-positive or SDC 2-positive or GPC 1-positive or GPC 3-positive CTC with an HSPG antibody or an SDC1 subtype antibody or an SDC2 subtype antibody or a GPC1 subtype antibody or a GPC3 subtype antibody in combination with immunomagnetic beads, using HSPG as a functional tumor molecular marker for sorting CTC that is relatively specific for gallbladder cancer and cholangiocarcinoma, thereby improving the sorting efficiency of the CTC of the biliary tract tumor, establishing a set of molecular typing scoring system of the CTC based on HSPG, obtaining a detection method for high specificity and high sensitivity of the biliary tract tumor, carrying out downstream RNA-seq and gene NGS detection based on the efficiently sorted CTC of the biliary tract tumor, obtaining tumor specific molecular information for improving the specificity, capturing tumor cells in blood through specific sorting antibody, and then the spider silk horse track of the genetic information carried by the released DNA is obtained and detected and analyzed, thus realizing the analysis method for detecting the DNA in time by using a non-invasive liquid biopsy mode.

Drawings

FIG. 1 is a schematic diagram of the formation process of the immunoliposome magnetic particle of the present invention.

FIG. 2 is a flow chart of the present invention for identifying CTC by immunomagnetic lipid particle sorting.

Detailed Description

The specific sorting, identifying and detecting method of CTC in serum of biliary tract tumor of the present invention is further described in detail below.

Example 1

A method for specifically sorting CTC in serum of biliary tract tumor comprises the following steps:

(2) sequentially carrying out sample treatment for removing plasma protein and plasma nucleic acid, removing red blood cells, carrying out layered centrifugation and removing white blood cells on the blood sample collected in the step (1), and specifically:

s4: and (3) removing white blood cells: centrifuging the anticoagulation centrifuge tube in S3 to obtain three layers of solution, gently sucking the uppermost 2 layers of solution into a new centrifuge tube, adding buffer solution, reversing and mixing uniformly, centrifuging to remove supernatant, adding buffer solution, and mixing uniformly to precipitate cells;

(3) washing the magnetic particles: sucking a proper amount of immune magnetic particles, namely HSPG antibody combined suspension liquid into an EP tube, standing, sucking and discarding the solution, adding a buffer solution, mixing uniformly, standing, discarding supernatant, repeating for 3 times, and then resuspending the magnetic particles to the original volume by using the buffer solution;

wherein the immunomagnetic particles combined with HSPG antibodies consist of the following components: iron oxide nanoparticles, cholestrol, GHDC, DOPC, HSPG;

(4) antibody incubation: slowly adding 10 mu L of immune magnetic particles into the 7.5mL sample treated in the step (2), uniformly pouring, adjusting the shaking speed of a shaking table, obliquely fixing the centrifugal tube on the shaking table, shaking at room temperature for 30min, and uniformly mixing once every 10 min;

(6) after standing for 10min, the clear solution was aspirated off, followed by the addition of 1mL of ddH₂O cleaning the magnetic ball;

(7) adding 100 mu LCF1 fixing solution for smear, and drying at 37 ℃;

Example 2

(3) washing the magnetic particles: sucking a proper amount of immune magnetic particles, namely mixed suspension of the antibody combined with the SDC1 subtype into an EP (EP) tube, standing, sucking and discarding the solution, adding a buffer solution, mixing uniformly, standing, discarding supernatant, repeating for 3 times, and then re-suspending the magnetic particles to the original volume by using the buffer solution;

wherein the immunomagnetic particles combined with the SDC1 subtype antibody consist of the following components: iron oxide nanoparticles, cholestrol, GHDC, DOPC, SDC 1;

(6) after standing for 10min, the clear solution was aspirated off, followed by the addition of 1mL of dd H₂O cleaning the magnetic ball;

(7) adding 100 mu LCF1 fixing solution for smear, and drying at 37 ℃;

Example 3

(3) washing the magnetic particles: sucking a proper amount of immune magnetic particles, namely mixed suspension of the antibody combined with the SDC2 subtype into an EP (EP) tube, standing, sucking and discarding the solution, adding a buffer solution, mixing uniformly, standing, discarding supernatant, repeating for 3 times, and then re-suspending the magnetic particles to the original volume by using the buffer solution;

wherein the immunomagnetic particles combined with the SDC2 subtype antibody consist of the following components: iron oxide nanoparticles, cholestrol, GHDC, DOPC, SDC 2;

(7) adding 100 mu LCF1 fixing solution for smear, and drying at 37 ℃;

(8) adding 4% paraformaldehyde dropwise, fixing for 8min, and preheating in water bath at 37 deg.C containing 2 XSSC buffer solution

10min；

(10) add 10. mu.L of fluorescent probe and coverslip, then place in the hybridization apparatus for hybridization at 76 ℃ for 10min and 37 ℃

Hybridizing for 1.5 h;

Example 4

(3) washing the magnetic particles: sucking a proper amount of immune magnetic particles, namely antibody suspension combined with GPC1 subtype, into an EP tube, standing, sucking and discarding the solution, adding a buffer solution, mixing uniformly, standing, discarding supernatant, repeating for 3 times, and then resuspending the magnetic particles to the original volume by using the buffer solution;

wherein the immunomagnetic particles combined with the GPC1 subtype antibody consist of the following components: iron oxide nanoparticles, cholestrol, GHDC, DOPC, GPC 1;

(7) adding 100 mu LCF1 fixing solution for smear, and drying at 37 ℃;

Example 5

(3) washing the magnetic particles: sucking a proper amount of immune magnetic particles, namely antibody suspension combined with GPC3 subtype, into an EP tube, standing, sucking and discarding the solution, adding a buffer solution, mixing uniformly, standing, discarding supernatant, repeating for 3 times, and then resuspending the magnetic particles to the original volume by using the buffer solution;

wherein the immunomagnetic particles combined with the GPC3 subtype antibody consist of the following components: iron oxide nanoparticles, cholestrol, GHDC, DOPC, GPC 3;

(7) adding 100 mu LCF1 fixing solution for smear, and drying at 37 ℃;

10min；

Hybridizing for 1.5 h;

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A method for specifically sorting CTC in biliary tract tumor serum is characterized in that: the method comprises the following steps:

(7) adding a CF1 fixing solution for smear, and drying at 37 ℃;

2. The method of claim 1, wherein the method comprises specific sorting of CTCs in serum of biliary tumors by: the processes of removing plasma protein and plasma nucleic acid, removing red blood cells, layering and centrifuging and removing white blood cells in the blood sample processing process in the step (2) are specifically as follows:

3. The method of claim 1, wherein the method comprises specific sorting of CTCs in serum of biliary tumors by: the immunomagnetic particles in the step (3) are combined with one of HSPG antibody, SDC1 subtype antibody, SDC2 subtype antibody, GPC1 subtype antibody and GPC3 subtype antibody.

4. The method of claim 3, wherein the method comprises specific sorting of CTC in serum from biliary tumors by: the immune magnetic particle combined with the HSPG antibody consists of the following components: iron oxide nanoparticles, cholestrol, GHDC, DOPC, HSPG.

5. The method of claim 3, wherein the method comprises specific sorting of CTC in serum from biliary tumors by: the immunomagnetic particles combined with the SDC1 subtype antibody consist of the following components: iron oxide nanoparticles, cholestrol, GHDC, DOPC, SDC 1.

6. The method of claim 3, wherein the method comprises specific sorting of CTC in serum from biliary tumors by: the immunomagnetic particles combined with the SDC2 subtype antibody consist of the following components: iron oxide nanoparticles, cholestrol, GHDC, DOPC, SDC 2.

7. The method of claim 3, wherein the method comprises specific sorting of CTC in serum from biliary tumors by: the immunomagnetic particles combined with the GPC1 subtype antibody consist of the following components: iron oxide nanoparticles, cholestrol, GHDC, DOPC, GPC 1.

8. The method of claim 3, wherein the method comprises specific sorting of CTC in serum from biliary tumors by: the immunomagnetic particles combined with the GPC3 subtype antibody consist of the following components: iron oxide nanoparticles, cholestrol, GHDC, DOPC, GPC 3.

9. The method of claim 1, wherein the method comprises specific sorting of CTCs in serum of biliary tumors by: in the step (4), 10 μ L of immunomagnetic particles are put into 7.5mL of sample and mixed by inversion.

10. A method for identifying and detecting CTC in biliary tract tumor serum is characterized by comprising the following steps: a method for extracting DNA based on circulating tumor cells by using a QIAGEN kit comprises the following specific steps:

(a) taking 10ml of the blood sample treated in the step (2), adding 2.5 times of Solutiona, reversing, uniformly mixing, centrifuging at 8000rpm for 5 minutes, removing supernatant, and repeating the steps for 1-2 times;

(f) adding 1000 mu L of Elutionbuffer, incubating at 60 ℃ for 10-15 minutes or overnight to dissolve DNA, blowing and beating uniformly by using a pipette or a disposable plastic pipette, and storing the DNA at-20 ℃;