CN112462058A

CN112462058A - Circulating nerve cell detection kit and detection method

Info

Publication number: CN112462058A
Application number: CN202011309903.XA
Authority: CN
Inventors: 丁显廷; 张瑜
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2020-11-20
Filing date: 2020-11-20
Publication date: 2021-03-09

Abstract

The invention belongs to the field of medical biotechnology research, and discloses a circulating nerve cell detection kit containing a fluorescence-labeled neuron specific antibody, a fluorescence-labeled leukocyte classical marker antibody and a cell nucleus fluorescent dye in the first aspect of the invention. Compared with the conventional detection means, the technical scheme disclosed by the invention has the advantages of high specificity, simplicity in detection and sampling, lower detection cost price and capability of realizing the technical effect of monitoring the blood sample in real time.

Description

Circulating nerve cell detection kit and detection method

Technical Field

The invention belongs to the field of medical biotechnology research, and relates to a circulating nerve cell detection kit and a detection method.

Background

In recent years, the trend of the age of stroke onset in China is obvious, which can seriously affect the development of socioeconomic development. It has been shown that stroke is closely related to many diseases, such as diabetes, hypertension and heart disease, and even genetic factors. Because the heterogeneity of the attack factors of the cerebral apoplexy is very high, how to conveniently, quickly and accurately diagnose the cerebral apoplexy has important significance for the prevention and the prognosis monitoring of the cerebral apoplexy. Ischemic stroke is currently diagnosed mainly based on clinical diagnosis and neuroimaging diagnosis, which depends largely on local availability and is difficult to detect at an early stage of ischemic stroke occurrence.

However, the currently available diagnostic and imaging diagnostic techniques have drawbacks. The computer tomography technology, the non-contrast computer tomography, the computer tomography angiography and the magnetic resonance imaging can not distinguish the acute ischemic stroke from the chronic ischemic stroke, the chronic hemorrhage including the micro-hemorrhage is difficult to detect, the capability of evaluating the subarachnoid space and the intracerebral hemorrhage is insufficient, the contrast agent used in the imaging process has corresponding renal toxicity, and the detection cost is expensive. The biomarker based on liquid biopsy is mainly derived from molecular level biomarkers in peripheral blood, plasma, serum and cerebrospinal fluid, and mainly comprises protein released by nerve cells after central nervous system diseases such as stroke and the like. Mainly comprises S100B protein, MMP-9 protein, interleukin 6, adhesion molecule and the like. These protein-level biomarkers require a certain latency period in the circulation to be detected. For example, the release of S100B is delayed and lacks specificity for stroke detection, and therefore, clinical use is limited. In addition, the detection time is one of the most critical factors in the diagnosis of the stroke. While microarrays can provide high throughput screening of thousands of genes, it takes about 11 hours to perform the analysis. In addition, special instruments and experienced personnel are required to perform the analysis, which is not conducive to clinical point-of-care testing. Research on central nervous system diseases such as cerebral arterial thrombosis focuses on protein and ribonucleic acid. Proteins and ribonucleic acids are released from nerve cells after ischemic brain injury and cross the blood-brain barrier into the peripheral blood. Damage to the blood-brain barrier and formation of cerebral edema are important factors in the development and progression of acute and chronic cerebral ischemic neurological dysfunction. The specific biomarker of ischemic stroke can remarkably promote the prediction of stroke, particularly the early stage of the occurrence of early stroke, and provide a certain supplementary effect on neuroimaging analysis.

For ischemic stroke, most of the research on biomarkers has focused on proteins and ribonucleic acids, which are released from nerve cells after ischemic brain injury and pass through the blood-brain barrier into the peripheral blood. The blood-brain barrier is a dynamic network that regulates the exchange of substances between the circulatory system and the parenchyma of the brain, while maintaining homeostasis in the central nervous system. Damage to the blood-brain barrier and formation of cerebral edema are important factors in the development and progression of acute and chronic cerebral ischemic neurological dysfunction. The specific biomarker of ischemic stroke can remarkably promote the prediction of stroke, particularly the early stage of stroke occurrence, and provide a certain supplementary effect on neuroimaging analysis. Therefore, there is a need for biomarkers that can accurately predict the occurrence and recurrence of acute ischemic stroke. Circulating cells, released into the circulation by the respective organs, produce molecular biomarkers (proteins, micro-RNAs and free nucleic acids) that can reveal key information about health and disease.

However, no specific biomarker which can accurately describe the occurrence and the development of the ischemic stroke on a cellular level is determined by the scholars at present, and how to reveal key information of health and diseases through the characteristic biomarker of the circulating nerve cells. Therefore, those skilled in the art are devoted to developing a circulating neural cell assay kit and assay method.

Disclosure of Invention

In order to detect circulating nerve cells in the nervous system and find and determine the circulating nerve cells by detecting the molecular biomarkers generated by the circulating nerve cells, the application discloses in a first aspect a circulating nerve cell detection kit, which comprises: the kit comprises CTC-enriched CD36 antibody, HBSS buffer solution of 2% FBS, density gradient centrifugate, erythrocyte lysate, cell rupture blocking solution, fluorescence-labeled neuron specific antibody, fluorescence-labeled leukocyte classical marker antibody, cell nucleus fluorescent dye, micropore array chip, standard substance containing circulating nerve cell marker and quality control substance containing positive control and negative control.

Further, the cell rupture blocking solution was a phosphate buffered saline containing 0.3% Triton-100, 5% sheep serum (NGS) and 0.05% Tween 20 (Tween-20).

Further, the fluorescently labeled neuron-specific antibody is a fluorescently labeled neuron-specific nucleoprotein antibody, also called NeuN fluorescently labeled antibody, the fluorescently labeled leukocyte classical marker antibody is an APC-labeled mouse anti-human CD45 antibody, also called CD45-APC antibody, and the nuclear stain is 4', 6-diamidino-2-phenylindole, Hoechst33342, Hoechst33258 or Hoechst 34580.

Further, the microwell array chip was blocked with 3% BSA.

The second aspect of the present invention provides a density gradient centrifugation kit for detecting circulating nerve cells on the basis of the first aspect of the present invention, wherein the density gradient centrifugation kit further comprises a density gradient centrifuge tube and a density gradient centrifugate.

Further, the detection method of the density gradient centrifugation kit for detecting the circulating nerve cells comprises the following detection steps:

a1, incubating 5 ml blood sample with 25-125 microliter of CTC-enriched CD antibody for 20 minutes at room temperature to obtain mixed solution L1;

a2, adding 15 ml of HBSS buffer solution containing 2% FBS into the mixed solution L1, and fully and uniformly mixing to obtain a mixed solution L2;

a3, adding 15 ml of density gradient centrifugate to the bottom of a density gradient centrifuge tube through a small hole in the middle of the density gradient centrifuge tube;

a4 and mixed solution L2 are transferred to a density gradient centrifuge tube along the tube wall of the density gradient centrifuge tube;

a5, after balancing, centrifuging for 20 minutes at 1200g to obtain a centrifugate L1;

a6, after centrifugation, transferring the centrifugate L1 at the upper layer into a 15 ml centrifuge tube, after re-balancing, centrifuging for 8 minutes at 600g to obtain a centrifugate L2;

a7 and centrifugate L2, and after supernatant is discarded, centrifugate L3 is obtained, 0.5 ml of erythrocyte lysate is added into the centrifugate L3, and the mixture is incubated for 5 minutes at room temperature;

centrifuging the mixture A8 and 300g for 5 minutes to obtain a centrifugate L4, discarding 400 microliters of supernatant of the centrifugate L4 to obtain a mixed solution L3, and resuspending the remaining 100 microliters of the mixed solution L3 and transferring the resuspended mixed solution L3 to an addressable micropore array chip which is sealed by 3% BSA in advance;

a9, fixing cells on a micropore array chip, breaking membranes, punching, sealing, incubating the NeuN antibody overnight at 4 ℃, removing the NeuN antibody by suction, fully washing the NeuN antibody by PBS, fully and uniformly mixing a secondary antibody marked with fluorescence, a CD45-APC antibody and DAPI, adding the mixture on the micropore array chip, incubating the mixture in the dark for 2 hours, and fully washing the mixture by PBS;

a10, scanning and imaging the micropore array chip by means of a microscopic imaging system such as a high content microscope or a laser confocal microscope.

Further, in step a10, the detection result is NeuN⁺/CD45^-/DAPI⁺The cells of (a) are circulating nerve cells.

The third aspect of the invention provides an immunomagnetic bead kit for detecting circulating nerve cells on the basis of the first invention, wherein the immunomagnetic bead kit further comprises CD45 modified immunomagnetic beads.

Further, the detection method of the immunomagnetic bead kit for detecting the circulating nerve cells comprises the following detection steps:

b1, adding 10 times volume of erythrocyte lysate into the blood sample, performing lysis at room temperature in a dark place, and centrifuging for 5 minutes at 300g to obtain mixed liquor Q1;

b2, discarding the supernatant of the mixed solution Q1, adding 5 ml of HBSS buffer solution to resuspend cells, and centrifuging for 5 minutes at 300g to obtain a mixed solution Q2;

b3, discarding the supernatant of the mixed solution Q2, adding 1 ml of HBSS buffer solution to resuspend cells, and counting the cells of the cell suspension;

b4, adding a CD45-APC antibody according to the antibody using proportion based on cell counting, fully and uniformly mixing, and incubating for 1 hour at room temperature in a dark place to obtain a mixed solution Q3;

b5, centrifuging the mixed solution Q3 for 5 minutes at 300g, discarding the supernatant, adding 5 ml of HBSS buffer solution for resuspension, and centrifuging the mixture for 5 minutes at 300g to obtain the final product; centrifugate S1;

b6, repeating the step B5 once, then adding 0.2 ml of HBSS buffer solution for resuspension, adding the immune magnetic ball modified with CD45, fully mixing, and incubating for 15 minutes in a dark place to obtain a mixed solution Q4;

b7 and mixed solution Q4 are added with 0.8 ml of HBSS buffer solution to be fully mixed, the mixture is placed on a magnetic frame to be kept stand for 10 minutes, and after all the cells combined with the CD45 modified immunomagnetic spheres are adsorbed at one side of the magnetic frame, cell suspension containing candidate cells is sucked into a new centrifugal tube;

b8, centrifuging 300g of the cell suspension obtained in the step B7 for 5 minutes, sucking and discarding 900 microliters of supernatant, and resuspending and transferring the remaining 100 microliters of cell-containing centrifugate S2 into an addressable micropore array chip which is blocked by 3% BSA in advance;

b9, fixing cells on the micropore array chip, breaking membranes, punching, sealing, incubating the NeuN antibody overnight at 4 ℃, removing the NeuN antibody by suction and fully washing the NeuN antibody by PBS, fully and uniformly mixing a secondary antibody marked with fluorescence and DAPI, adding the mixture on the micropore array chip, incubating the mixture for 2 hours in a dark place, and fully washing the mixture by PBS;

and B10, scanning and imaging the micropore array chip by means of a microscopic imaging system such as a high content microscope or a laser confocal microscope.

Further, in step B10, the detection result is NeuN⁺/CD45^-/DAPI⁺The cells of (a) are circulating nerve cells.

The invention has the beneficial effects that:

1. the sampling is simple, only a blood sample needs to be collected, the circulating nerve cells can be separated, the circulating nerve cells are detected and analyzed, the monitoring of clinical prognosis can be carried out based on the number of the detected cells, and meanwhile, the circulating nerve cells can provide regional information of brain injury to a certain extent.

The blood sample collection device can be used for monitoring in real time, and can collect blood samples of patients at different time points of clinical monitoring at any time for detection and analysis, so that the effect of real-time monitoring is realized.

2. High specificity, and adopts the specific marker NeuN of neuron cell and the mark of leucocyteMarker CD45 and nuclear marker DAPI, NeuN in cells detected⁺/CD45^-/DAPI⁺The cells of (a) are considered to be circulating nerve cells, and because NeuN is a classical marker unique to neurons, the specificity of detection is higher.

3. The detection cost price is lower.

Drawings

FIG. 1 is a schematic diagram of a detection process of circulating nerve cell detection by density gradient centrifugation;

FIG. 2 is a schematic view of a detection process of circulating nerve cells by immunomagnetic beads;

FIG. 3 is a schematic diagram of an image of the number 21 region of a microwell array chip loaded with a sample cell suspension in a preferred embodiment in the light field;

FIG. 4 is a schematic diagram of immunofluorescence imaging of the 21 st encoded region of the microwell array chip with the sample cell suspension loaded thereon according to the preferred embodiment;

FIG. 5 NeuN in the sample of the preferred embodiment⁺/CD45^-/DAPI⁺Cells of (3) and NeuN^-/CD45⁺/DAPI⁺A schematic representation of a cell image of (a);

FIG. 6 is a schematic diagram illustrating a distribution of NeuN signal values in a preferred embodiment;

FIG. 7 is a schematic illustration of a bright field image of a portion of a cell of interest in a sample according to a preferred embodiment.

Detailed Description

For the convenience of understanding, the circulating neural cell markers, the identification method, the detection kit and the application are described below with reference to examples, which are only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1

Density gradient centrifugation

As shown in fig. 1, the method for separating and identifying the circulating nerve cells in the blood sample mainly comprises the following specific steps:

a) density gradient centrifugation: the patient peripheral blood samples were incubated with the corresponding volume of antibody and subjected to density gradient centrifugation.

b) Cell suspension was added to the chip: the cell suspension containing the cells of interest after density gradient centrifugation is loaded onto a microwell array chip.

c) Cell fixation, membrane rupture and staining: and (3) fixing and membrane rupture are carried out on the cells on the chip, and then staining treatment is carried out to identify the circulating nerve cells, wherein staining agents are a certain concentration of a fluorescence labeled neuron specific antibody NeuN antibody substance or a primary antibody of NeuN, a secondary antibody labeled with a fluorescence label, an antibody CD45 aiming at white blood cells, a dye DAPI of cell nucleus and the like.

d) Cleaning and imaging: and (3) fully washing the stained cells, then carrying out fluorescence analysis to determine cells which are positive to NeuN positive CD45 negative DAPI, and preliminarily judging the cells as the circulating nerve cells.

Wherein the fluorescently labeled antibody substance is an antibody directly labeled with fluorescein or other fluorescent substances (such as quantum dots), or a combination of unlabeled primary antibody and fluorescein or other fluorescent substance-labeled secondary antibody.

In this embodiment, the threshold for NeuN positivity is the mean of all or a portion of the leukocyte NeuN signal values in the sample plus five times the standard deviation.

In order to reduce or eliminate the interference of some non-cellular impurities (e.g. cell debris, gas bubbles, non-cellular particles, etc.) introduced into the test sample or during the treatment process, which have an adsorptive effect on the fluorescently labelled antibody species of NeuN, on the fluorescence analysis, particular embodiments of the method of the invention further comprise staining the sample cells with a nuclear dye, such as a nuclear fluorescent dye (preferably selected from the group consisting of 4', 6-diamidino-2-phenylindole (DAPI) or Hoechst series staining), simultaneously, prior or subsequent to the NeuN staining. In this case, a NeuN-positive cell which is positive for nuclear staining at the same time can be initially determined as a circulating nerve cell.

Example 2：

Immunomagnetic bead method

As shown in fig. 2, the method for separating and identifying the circulating nerve cells in the peripheral blood of the central nervous system disease mainly comprises the following specific steps:

1. and (3) cracking red blood cells: erythrocytes in the peripheral blood of a patient are lysed with erythrocyte lysate.

2. Negative selection: and adding immunomagnetic beads modified with CD45 into the cell suspension after red cracking, fully incubating, and screening by a magnetic frame.

3. Cell suspension was added to the chip: after negative selection, the cell suspension in the centrifuge tube is loaded onto the microwell array chip after the centrifuge tube is maintained in position on the magnetic rack to extract the cell suspension.

4. Cell fixation, membrane rupture and staining: and (3) fixing and membrane rupture are carried out on the cells on the chip, and then staining treatment is carried out to identify the circulating nerve cells, wherein staining agents are a certain concentration of a fluorescence labeled neuron specific antibody NeuN antibody substance or a primary antibody of NeuN, a secondary antibody labeled with a fluorescence label, an antibody CD45 aiming at white blood cells, a dye DAPI of cell nucleus and the like.

5. Cleaning and imaging: and (3) fully washing the stained cells, then carrying out fluorescence analysis to determine cells which are positive to NeuN positive CD45 negative DAPI, and preliminarily judging the cells as the circulating nerve cells.

Example 3：

Identifying and analyzing the circulating nerve cells in the peripheral blood of the cerebral apoplexy patient for monitoring the clinical prognosis of the cerebral apoplexy patient.

1. 5 ml of patient peripheral blood (patient-known) was incubated with 75 microliters of CTC-enriched antibody (including CD36 antibody) for 20 minutes at room temperature.

2. 15 ml of Hank's Balanced Salt Solution (HBSS) containing 2% Fetal Bovine Serum (FBS) was added to the blood in 1 and mixed well.

3. 15 ml of density gradient centrate was added to the bottom of the centrifuge tube through a small hole in the middle of the centrifuge tube.

4. The well-mixed patient blood from step 2 is transferred along the tube wall to a density gradient centrifuge tube in 3, preferably without disturbing the density gradient centrifugate at the bottom of the tube.

5. Under the precondition of balancing, 1200g are centrifuged for 20 minutes.

6. After centrifugation, the supernatant was transferred to a 15 ml centrifuge tube and centrifuged at 600g for 8 minutes with trimming.

7. After centrifugation, the cells were discarded, 0.5 ml of erythrocyte lysate was added, and incubated at room temperature for 5 minutes.

8. After centrifugation at 300g for 5 minutes and discarding 400. mu.l of the supernatant, the remaining 100. mu.l of the cell-containing liquid was resuspended and transferred to an addressable microwell array chip previously blocked with 3% BSA, which in this example comprises a total of 100 numbered regions, and a total of about 3 ten thousand addressable microwells for holding and positioning cells, wherein each microwell has a diameter of 30 μm, so that the cells were uniformly distributed on the chip and sunk into the microwells of the chip, as shown in FIGS. 3 and 4, and the sample was in a single cell dispersed state as an image of one numbered region of the microwell chip to which the sample cell suspension was loaded.

9. 100 microliters of 4% Paraformaldehyde (PFA) was added to the chip and fixed at room temperature for 30 minutes, and 130 microliters of Phosphate Buffered Saline (PBS) was used to wash the chip 3 to 5 times.

10. The PFA-fixed cells were subjected to membrane rupture and blocking with 100. mu.l of phosphate buffer containing 0.3% Triton-100, 5% goat serum (NGS), 0.05% Tween 20(Tween-20), incubated at room temperature for 1 hour, and the chips were washed 3 to 5 times with 130. mu.l of Phosphate Buffer (PBS).

11. The primary antibody to NeuN antibody was added to the chip, and after overnight incubation at 4 deg.C, the chip was washed 3-5 times with 130. mu.l of Phosphate Buffered Saline (PBS).

12. 100 microliters of a solution of a secondary antibody labeled with fluorescein FITC, CD45-APC, and DAPI was added to the chip, incubated at room temperature for 2 hours in the dark, and the chip was washed 3-5 times with 130 microliters of Phosphate Buffered Saline (PBS).

13. Scanning and imaging the micropore array chip containing the circulating nerve cells by means of a microscopic imaging system such as a high content microscope or a laser confocal microscope, wherein the NeuN⁺/CD45^-/DAPI⁺Is defined as a circulating neural cell. Wherein the threshold value of NeuN positivity is the average value of the NeuN signal values of all or part of the leucocytes on the chip plus five times of standard deviation. The DAPI positive judgment standard is that the DAPI positive judgment standard is positive if the DAPI positive judgment standard is chromogenic.

FIG. 5 shows the cells detected in the samples of examples, in which the top two rows of cells, NeuN-FITC positive, CD45-APC negative, DAPI positive, are circulating nerve cells. The bottom row of cells, NeuN-FITC negative, CD45-APC positive, DAPI positive, are leukocytes in peripheral blood.

FIG. 6 shows the distribution of the cells in the number 0-99 regions in the sample of example 1, which were statistically derived from the NeuN signal and the CD45 signal, wherein the cells above the NeuN threshold were determined to be the target cells. Fig. 7 shows bright field images of a portion of the target cells in the sample of example 1, where the cells are all larger and the nuclei are all more distinct, with a scale of 5 microns.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A circulating nerve cell detection kit, characterized in that, the detection kit comprises: the kit comprises CTC-enriched CD36 antibody, HBSS buffer solution of 2% FBS, density gradient centrifugate, erythrocyte lysate, cell rupture blocking solution, fluorescence-labeled neuron specific antibody, fluorescence-labeled leukocyte classical marker antibody, cell nucleus fluorescent dye, micropore array chip, standard substance containing circulating nerve cell marker and quality control substance containing positive control and negative control.

2. The circulating neural cell detection kit of claim 1, wherein the fluorescently labeled neuron-specific antibody is a fluorescently labeled neuron-specific nucleoprotein antibody, also known as a NeuN fluorescently labeled antibody, the fluorescently labeled leukocyte classical marker antibody is an APC-labeled mouse anti-human CD45 antibody, also known as a CD45-APC antibody, and the nuclear stain is 4', 6-diamidino-2-phenylindole, Hoechst33342, Hoechst33258, or Hoechst 34580.

3. The circulating neural cell detection kit of claim 1, wherein the microwell array chip is blocked with 3% BSA.

4. The circulating neural cell assay kit of claim 1, wherein the cell rupture blocking solution is a phosphate buffered saline containing 0.3% Triton-100, 5% sheep serum (NGS) and 0.05% Tween 20 (Tween-20).

5. The density gradient centrifugation kit for the circulating nerve cell detection kit according to claim 2, wherein the density gradient centrifugation kit further comprises a density gradient centrifuge tube and a density gradient centrifugate.

6. The method of detecting a density gradient centrifugation kit of claim 5, wherein the detecting step comprises:

a9, fixing cells on a micropore array chip, breaking membranes, punching, sealing, incubating the NeuN antibody overnight at 4 ℃, removing the NeuN antibody by suction, fully cleaning the NeuN antibody by PBS, adding a fluorescence labeled secondary antibody, fully mixing a CD45-APC antibody and DAPI, adding the mixture on the micropore array chip, incubating the mixture in a dark place for 2 hours, and fully cleaning the mixture by PBS;

7. The method for detecting a density gradient centrifugation kit according to claim 6, wherein in the step A10, the detection result is NeuN⁺/CD45^-/DAPI⁺The cells of (a) are circulating nerve cells.

8. The immunomagnetic bead kit of the circulating nerve cell detection kit of claim 2, wherein the immunomagnetic bead kit further comprises CD 45-modified immunomagnetic beads.

9. The method of claim 8, wherein the step of detecting comprises:

b4, 1X10 based on cell count⁷Adding the CD45-APC antibody into the cells according to the proportion of 20 microliter, fully and uniformly mixing, and incubating for 1 hour at room temperature in a dark place to obtain a mixed solution Q3;

b7 and mixed solution Q4 are added with 0.8 ml of HBSS buffer solution to be fully mixed, the mixture is placed on a magnetic frame to be kept stand for 10 minutes, and after all the cells combined with the CD45 modified immunomagnetic spheres are adsorbed at one side of the magnetic frame, cell suspension containing candidate cells is sucked into a centrifuge tube;

10. The method for detecting an immunomagnetic bead kit of claim 9, wherein in the step A10, the detection result is NeuN⁺/CD45^-/DAPI⁺The cells of (a) are circulating nerve cells.