CN114563571B

CN114563571B - Application of CKAP4 reagent in detection specimens and bladder cancer detection kit

Info

Publication number: CN114563571B
Application number: CN202210065609.1A
Authority: CN
Inventors: 叶茂; 谭蔚泓; 孙星; 李霞辉
Original assignee: Hunan University
Current assignee: Hunan University
Priority date: 2022-01-20
Filing date: 2022-01-20
Publication date: 2025-01-28
Anticipated expiration: 2042-01-20
Also published as: CN114563571A

Abstract

The invention discloses application of a CKAP4 reagent in a detection sample and a bladder cancer detection kit. CKAP4 is not only highly expressed in bladder cancer cell lines and their excreted exosomes, but more importantly, CKAP4 is clinically significantly highly expressed in urinary exosomes of bladder cancer patients, but has significantly low expression levels in urinary system cancers such as prostate cancer, renal cell carcinoma, etc., urinary system inflammation, and urine exosomes of healthy donors. Therefore, the detection means based on the urine exosome CKAP4 is a non-invasive liquid biopsy method which is low in cost, simple and capable of meeting the requirement of multiple detection, and is particularly suitable for diagnosing and recurrence monitoring of patients with bladder cancer.

Description

Application of CKAP4 reagent in detection specimen and bladder cancer detection kit

Technical Field

The invention belongs to the technical field of tumor molecular biology, and relates to application of a CKAP4 reagent in a detection sample and a bladder cancer detection kit.

Background

Bladder cancer is a malignant urinary system disease that occurs in the bladder epithelium, and data in 2020 shows that the incidence and mortality rate of bladder cancer are all the first 10 worldwide, with 57.3 thousands of new cases and 21.3 die ten thousand deaths deaths each year. Of these, approximately 75% are non-myogenic invasive bladder cancers and 25% are myogenic invasive bladder cancers. However, whether or not myogenic layer infiltration occurs, an important feature of bladder cancer is its high recurrence, and metastasis of bladder cancer can cause rapid progression of the disease and cause death of the patient in a short period of time. Therefore, establishing a rapid, simple, inexpensive method that can meet the high frequency detection requirements is a key technology to cope with bladder cancer recurrence and improve the therapeutic effect.

In the existing detection method, cystoscope tissue biopsy is a gold standard for clinical diagnosis, the technology is that cystoscope is inserted into the bladder through the urethra, the pathological changes in the bladder and the urethra are directly observed, and suspected focal tissues are cut off for the invasive detection method for pathological diagnosis, so that the accuracy is high. However, the invasive examination method not only needs a doctor to have abundant operation experience, but also has a plurality of complications, is easy to miss flat urothelial carcinoma in situ, has low acceptance of patients, and is difficult to meet the requirement of large-scale popularization. Imaging examination based on ultrasound, CT, MRI and other techniques is a widely used detection means in clinic. For bladder cancer, especially early non-invasive bladder cancer or recurrent micro focus after operation, the imaging detection means often cause misdiagnosis and missed diagnosis phenomena due to the limitation of instrument resolution, and has larger limitation. In addition, the performance indexes such as sensitivity and specificity of the existing urine-based noninvasive detection technologies such as urine abscission cytology, nuclear matrix protein 22 (NMP 22) and chromosome Fluorescence In Situ Hybridization (FISH) are difficult to meet the requirements of clinical diagnosis, and particularly, the detection sensitivity of the existing urine-based noninvasive detection technologies for low-level bladder cancer is poor, so that early screening of bladder cancer cannot be realized.

The exosomes are microvesicles secreted by cells and have the size of 30-100nm, carry biological information in a plurality of cells, and can accurately reflect the molecular biological characteristics of cells from which the exosomes are derived. Because of the size limitation of glomerular filtration, exosomes in blood cannot directly enter the urinary system, so that exosomes in urine are derived from urinary system cells, and interference of exosomes secreted by other systemic cells can be effectively eliminated. Research shows that compared with normal cells, cancer cells have stronger exosome secretion capacity due to vigorous proliferation, and can secrete a larger amount of exosomes containing cell biological information into the extracellular environment. In terms of separation and purification, due to the size effect of an exosome, inorganic salts with small molecular weight, biological small molecules, impurity proteins, exfoliated cells with large molecular weight and other interfering impurities can be greatly excluded, so that bladder cancer is taken as a disease which is caused in the urinary system, and the noninvasive detection of the bladder cancer is realized through the biological characteristics of the exosome of urine of a patient, so that the method is a novel molecular detection means with great prospect.

Currently, TACSTD, AAT, filamin-A and H2B1K are found to have higher content in tumor-derived exosome samples in the aspect of exosome biomarker research, but the signals are found to be poor through detection by a specific antibody blotting technology, and the statistical difference between each group of patients and a control sample can not be achieved, and the defects of detection sensitivity are added, so that the clinical application results are difficult to convert. Therefore, the urine exosome biomarker which can be used for the clinical detection of bladder cancer is found to have obvious application advantages and market demands.

Disclosure of Invention

The invention discloses a protein molecule CKAP4 with bladder cancer specificity, which is not only highly expressed in a bladder cancer cell line and an exosome thereof, but also is specifically and highly expressed in the exosome of urine of a bladder cancer patient. The invention also discovers that the CKAP4 regulates the mechanical property of the cell surface through the exosome, thereby promoting the migration of cells and the metastasis in the mouse body, and emphasizes the important role of the CKAP4 in the bladder cancer exosome.

The primary object of the invention is to provide an application of CKAP4 reagent in a detection sample for preparing a bladder cancer diagnosis preparation, wherein the sample comprises at least one of tissues, cells, cell exosomes, blood, urine and urine exosomes.

Further, CKAP4 is expressed in samples from patients with bladder cancer in higher amounts than healthy persons, urinary system inflammation, or other cancer patients.

Further, the CKAP4 reagent in the test sample comprises a reagent for detecting the content of CKAP4 protein in the sample.

Further, the CKAP4 reagent in the detection sample also comprises a reagent for extracting CKAP4 protein in the sample.

Further, the specimen comprises at least one of urine and urine exosomes, in particular urine exosomes.

The second object of the invention is to provide a bladder cancer detection kit, which comprises a reagent for detecting the CKAP4 protein content in a specimen.

Further, the kit also comprises a reagent for extracting CKAP4 protein from the specimen.

The specimen comprises at least one of tissues, cells, cell exosomes, blood, urine and urine exosomes.

The invention also provides application of CKAP4 in research of regulating and controlling mechanical properties of cell surfaces through transportation of exosomes and maintaining high hardness gradient of the cell surfaces from center to edge. This feature can significantly promote metastasis of bladder cancer.

Clinically, the invention collects morning urine samples of bladder cancer patients with different grades before and after treatment, adopts a super-high speed centrifugation method to extract exosomes, verifies the expression condition of CKAP4 in the exosomes by werstern blot, determines that the specificity of the CKAP4 in the urine exosomes of the bladder cancer patients is high, obviously reduces the expression level in the samples after tumor is excised by operation, and has low expression level in the urine exosomes of urinary system inflammation patients and healthy human donors. Among the 32 samples according to the present invention, the sensitivity, specificity and accuracy of detection based on exosome CKAP4 were 93.8% (15 samples were detected in 16 bladder cancer positive patients), 100% (no CKAP4 expression was detected in 16 non-bladder cancer negative samples), and 96.8% (31 samples in all 32 samples were in accordance with the detection expectation) (see fig. 7), respectively, and the excellent selectivity and sensitivity thereof were suitable for clinical detection standards.

The exosome protein molecule for detecting bladder cancer also comprises the following application:

(1) Based on clinical detection means of exosome CKAP4 in urine of patients with bladder cancer, such as detection test paper, kit, device, instrument, etc.

(2) Use in the preparation of a carrier reagent for the treatment of bladder cancer.

(3) Use in studying the differentiation of bladder cancer cells from normal cells.

(4) Application in researching bladder tumor in-vivo imaging.

Compared with the prior art, the invention has the advantages that:

1. the detection sensitivity, specificity and accuracy based on the exosome CKAP4 in urine of the patient with bladder cancer are 93.8%, 100% and 96.8% respectively, which are far greater than the existing detection means based on free protein and exfoliated cells;

2. The exosomes collected by the invention can come from bladder cancer cells at different disease sites, can reflect the overall condition of the disease sites of a patient, and avoid the possible missed detection condition in the detection methods such as cystoscope and the like;

3. The detection means can well avoid the influence of larger exfoliated cells, precipitates and metabolites in clinical samples, and can avoid the influence of free impurity proteins which cannot be removed;

4. the detection means based on the exosome protein CKAP4 can be used for primary disease detection, follow-up detection and other applications.

The advantages enable the exosome CKAP4 to have important potential in the aspects of diagnosing and targeted treatment of bladder cancer as a bladder cancer specific recognition molecular probe.

Drawings

FIG. 1 expression of CKAP4 in bladder cancer tissue;

a) CKAP4 is highly expressed in patients with bladder cancer compared to normal tissue, wherein BLCA (N-) represents bladder cancer without lymph node metastasis, BLCA (N+) represents bladder cancer with lymph node metastasis, NMIBC represents non-invasive bladder cancer, MIBC represents invasive bladder cancer;

b) Of 176 samples, the positive samples of CKAP4 in the bladder cancer patients (samples with CKAP4 expression score higher than that of normal tissue CKAP 4) are 165, and the positive rate is 93.75%;

c) High expression of CKAP4 is related to bladder cancer progress, NMIBC is non-invasive bladder cancer, MIBC is invasive bladder cancer, P is less than 0.05, and as shown in the figure, CKAP4 expression amount increases with increasing bladder cancer wettability;

d) CKAP4 expression is related to metastasis of bladder cancer, N0 is not metastasized, N1-N2 is metastatic of bladder cancer, P is less than 0.05, and CKAP4 expression increases with increasing metastasis degree of bladder cancer lymph node as shown in the figure;

e) Forest charts assess risk factors associated with bladder cancer progression as shown, infiltration and metastasis of bladder cancer are more significant risk factors than age, sex.

FIG. 2 high expression of CKAP4 in bladder cancer cells and their secreted exosomes;

a) Western blot of CKAP4 expression in bladder cancer 5637 cells and normal cell line SV-HUC-1, wherein GAPDH is an internal reference;

b) The quantitative analysis of Western blot in FIG. 2 a) by using the principle of antigen-antibody specific recognition and binding, transferring the protein to PVDF membrane, recognizing and binding to CKAP4 by using Anti-CKAP4 specific antibody, and detecting and quantitatively analyzing the CKAP4 by using chemiluminescence method. As can be seen, CKAP4 is highly expressed in the cell line 5637 of bladder cancer compared to the normal cell SV-HUC-1;

c) Transmission electron microscopy of exosomes in 5637 cells and SV-HUC-2 cells isolated from cell culture supernatant, scale 100nm;

d) Particle size analysis of 5637 and SV-HUC-1 exosomes;

e) Western blot of CKAP4 expression in 5637 and SV-HUC-1 exosomes;

f) From FIG. 2 e), it is evident that CKAP4 is highly expressed in the exosomes secreted by the cells of bladder cancer cell line 5637, but expressed in the exosomes of normal cell SV-HUC-1 in a lower amount.

G-h) marking the expression of CKAP4 on the surface of the exosome with AuNPs modified by CKAP4 aptamer;

g) Scale 100nm, quantitative analysis h), P <0.0001.

FIG. 3 differential expression proteins for CKAP4 5637 and SV-HUC-1 exosomes;

a) The results show that, compared with the SV-HUC-1 exosome protein from normal cells, CKAP4 is the protein with high expression in bladder cancer exosome, and the expression difference is the protein with the maximum expression specificity of bladder cancer cells;

b) Protein mass spectrum data of CKAP4, wherein the data show peptide fragment information characteristic of CKAP 4.

FIG. 4 CKAP4 in intracellular and exosomes and CKAP4 promotes changes in cell surface mechanical properties;

wherein OE represents Overexpression, i.e., overexpressing CKAP4, shNC represents a control group for knockdown experiments, shCKAP represents knockdown CKAP4;

a) Schematic representation of atomic force probe measurement of cell surface hardness;

b) Force curve characteristics of the degree of softness of the cell surface;

c) Cell morphology (up) and surface softness (down) changes after bladder cancer 5637 cells knockdown or overexpress CKAP 4;

d) A curve of the degree of softness of the cell surface in the direction indicated by the arrow;

e) The statistics of FIG. 4 d), wherein each group contains greater than 15 cells, using student's t test for statistics with P <0.01 and P <0.0001, as seen in figures c-e, CKAP4 of bladder cancer cells maintains a high gradient of cell surface hardness from center to edge, decreasing gradient and increasing gradient when CKAP4 knocks down;

f) Exosome CKAP4 enhances the receptor cell surface hardness gradient;

g-h) a curve of the change in surface hardness of 5637 cells (g) and SV-HUC-1 cells (h) in the direction indicated by the arrow;

The statistical difference between the cell mechanical properties of i-j) 5637 cells (i) and SV-HUC-1 cells (j) was found to be at least 15 cells per group, using student's t test with P <0.05 with P <0.0001, ns without significant differences, as seen in figures f-j, with the effect of CKAP4 on bladder cancer exosomes, the hardness gradient was increased on both 5637 cells and SV-HUC-1 cells, whereas the exosomes alone did not significantly alter the cell surface mechanical properties.

FIG. 5 exosome CKAP4 enhances the motility of bladder cancer cells and enhances cell migration;

No Exo is added with No exosome, shNC Exo is added with exosome extracted from control group cells in the knockdown experiment, shCKAP Exo is added with exosome extracted from knockdown CKAP4 cells;

5637shNC a control group for knockdown experiments in 5637 cells, 5637shckap4 expression of CKAP4 in 5637shckap4:5637 cells;

a) The promotion of cell motility by CKAP4 and exosome CKAP4 in cells, each circle represents 10min, scale 20 μm, the motility of SV-HUC-1 cells with low expression of CKAP4 is weaker than that of 5637 cells with high expression of CKAP4, the motility of cells after the CKAP4 in the 5637 cells is knocked down is weakened, then exosomes containing CKAP4 and not containing CKAP4 are respectively added in a control group and an experimental group, and the cell motility of the experimental group is found to be enhanced after the exosomes containing CKAP4 are added;

b) The method comprises the steps of tracking a cell track, analyzing the cell motion track, wherein the motion range of SV-HUC-1 cells which are low in CKAP4 expression is smaller than that of 5637 cells which are high in CKAP4 expression, knocking down the CKAP4 in the 5637 cells, reducing the cell motion range, and then adding exosomes containing CKAP4 and not containing CKAP4 into a control group and an experimental group respectively, wherein the cell motion range of the experimental group is increased after adding the exosomes containing CKAP 4;

c) Statistical data of promotion of cell motility by intracellular CKAP4 and exosome CKAP4, more than 50 cells were counted in each group, using student's t test with P <0.01 and P <0.0001 ns, without significant difference, statistical analysis of the motility, i.e. the motility distance, of each cell and under the conditions of FIG. 4 a), found that there was a significant difference between the motility distance of exosomes with CKAP4 added and exosomes without CKAP4 added in the control group, whereas in the experimental group the difference was more pronounced, and the same result was seen in SV-HUC-1 with low expression of CKAP 4;

d) In the control group, the exosomes without CKAP4 are not added or added, and the wound healing capacity, namely the cell migration capacity, of the exosomes without CKAP4 is poorer than that of a group of exosomes with CKAP4, and the migration capacity of the cells is weakened after the CKAP4 is knocked down in the experimental group, and the migration capacity is recovered after the exosomes with CKAP4 are added;

e) The statistical analysis in fig. 5 d) using student's t test with P <0.01 with P <0.0001, ns, no significant differences in the migration ability of cells without exosomes added to those with CKAP4 exosomes, and no significant differences in the migration ability of cells without exosomes added to those with CKAP4 exosomes in the control group;

f) In the control group, exosomes without CKAP4 are not added or added, and the cell transfer capacity is poorer than that of a group of exosomes containing CKAP4, and the cell transfer capacity is weakened after the CKAP4 is knocked down in the experimental group, and the cell transfer capacity is recovered after the exosomes containing CKAP4 are added;

g) Statistical analysis in fig. 5 f) using student's t test, × P <0.001, ns, no significant difference. In the control or experimental groups, there was a significant difference in the ability of cells to transfer after addition of exosomes containing CKAP4 compared to the group without exosomes.

FIG. 6 effect of exosome CKAP4 on metastasis of bladder cancer cells in mice;

a) Schematic representation of a metastasis model of bladder cancer cells in mice;

b) Schematic of each set of information for cell and exosome effects;

c) The high expression of CKAP4 promotes the bladder cancer cells to colonize the liver, the HE staining, the Ki67 staining and the CKAP4 staining are obviously enhanced at the expression site of the metastatic focus, the transfer of the liver is further enhanced by the CKAP4 under the action of exosomes, and the exosomes without CKAP4 do not obviously change the liver transfer of the bladder cancer cells.

D-f) statistical analysis of bladder cancer cells at the site of liver metastasis;

d) Number of tumor nodes;

e) Ki-67 index;

f) The expression of CKAP4 adopts student's t test, P <0.05, P <0.01, P <0.001, P <0.0001, and the data in the graph d-f) show that the high expression of the CKAP4 in the bladder cancer cells promotes liver metastasis, the transfer capacity of the bladder cancer cells is enhanced under the action of the exosome CKAP4, and the exosome which does not express the CKAP4 has no obvious promotion effect on the liver metastasis of the bladder cancer cells.

FIG. 7 expression of CKAP4 in urine exosomes from patients with bladder cancer;

a) Representative western blots of CKAP4 expression in urine exosome samples from healthy Humans (HD), bladder cancer (high grade hBLCA; low grade lBLCA), post-bladder cancer (AS), prostate cancer (PRAD), kidney cancer (KIRC), benign tumor of the bladder (bBL) patients;

b) Thermographic analysis of the relative amounts of CKAP4 in all urine exosome samples tested, wherein non-bladder tumors such as prostate cancer, renal cancer, etc. are collectively referred to as non-bladder cancers (nBLCA);

c) Statistical analysis of the relative content of CKAP4 in all urine exosome samples tested, P <0.05, P <0.01.

Detailed Description

The following examples are intended to further illustrate the invention, but not to limit it.

1. Materials and instruments

1. Material

1.1 Cell lines

5637 (Human bladder cancer cells), SV-HUC-1 (human bladder epithelial immortalized cells).

1.2 Medium, serum

DPBS, serum-free 1640, serum-free DMEM, PS, trypsin, zeta serum (ThermoFisher Scientific).

1.3 Antibodies

A rabbit anti-CKAP 4 polyclonal antibody (Biotechnology (Shanghai) Co., ltd., D261564), CD81 antibody (D-4) sc-166028, CD63 (E-12) sc-365604, alix (1A 12) sc-53540 (SANTA CRUZ BIOTECHNOLOGY), murine secondary antibody, and rabbit secondary antibody (Merck).

1.4 Clinical samples

Clinical samples were all from Yu Xiangya second affiliated hospital urological department, healthy human samples were derived from volunteer donations, and all were in accordance with biological ethics.

1.5 Other reagents

ECL chemiluminescent solution (Advansta), 1.5M Tris-HCl pH 8.8, 1.0M Tris-HCl pH6.8 and 10% SDS (Shanghai Weiao Biotechnology Co., ltd.).

2. Instrument for measuring and controlling the intensity of light

A low temperature ultra-high speed centrifuge and associated ultra-high speed centrifuge tube (Beckman), a disposable needle filter 0.22um33mm (Millipore), a centrifugal concentrator (LABCONCO), a programmed five-stage metal bath (Tiangen), a vertical electrophoresis apparatus, an electrophoresis tank and other electrophoresis fittings, and a Bere Chemiedoc XRS+ chemiluminescent gel imaging system (Biorad).

EXAMPLE 1 immunohistochemistry

Tissue microarrays containing normal, malignant and metastases were purchased from Avilabio (western An, china, DC-Bla 11021). The slide was immersed in a sodium citrate antigen recovery solution (pH 6.0). After blocking endogenous peroxidase and serum with 3%H ₂O₂, sections were incubated overnight with primary antibodies to CKAP4 at 4 ℃ and labeled with HRP secondary antibody for 50 minutes at room temperature. Finally, the sections were developed in freshly prepared DAB chromogenic reagent until the nuclei were brown-yellow under the microscope, and then counterstained with hematoxylin staining solution.

The expression levels of CKAP4 in the tissue microarrays were blindly examined and scored by two independent experienced pathologists and averaged percent values. CKAP4 expression was scored according to the ratio and intensity of cytoplasmic and nuclear staining as 0-10% score 0, 11-50% score 1, 50-75% score 2, and over 75% score 3. Hematoxylin stained nuclei appear blue. Positive cells cultured with DAB reagent had a brown-yellow nucleus. The results are shown in FIG. 1.

Example 2. Collection of exosomes in cell samples using gradient centrifugation.

The experiment comprises the following specific steps:

(1) Resuscitation 5637 (human bladder cancer cells), SV-HUC-1 (human bladder epithelial immortalized cells). After the cells proliferated to logarithmic growth phase, they were passaged to a very large dish. Cells in the oversized dish grew to 60% and the culture supernatant was collected and passaged. And repeatedly collecting the cell culture medium supernatant to a volume of 300-500 mL.

(2) Transfer the culture supernatant to a 50mL centrifuge tube, 4000rpm,30min,4 ℃, collect and transfer the transfer supernatant to a new 50mL centrifuge tube.

(3) Disposable needle filter 0.22um 33mm filtration and collection of supernatant.

(4) The supernatant was dispensed into Beckman ultracentrifuge tubes (55654), and the low temperature, ultra high speed centrifuge (Beckman_L-100 XP) was programmed to rotate at 110000g,1h,4 ℃.

(5) The supernatant was discarded and the pellet was resuspended with pre-chilled DPBS and the low temperature ultra high speed centrifuge program was designed to 110000g,1h,4 ℃.

(6) The supernatant was discarded, the exosomes were resuspended with residual liquid and thoroughly collected with pre-chilled 200-400 ul DPBS.

(7) The collected exosomes were concentrated to a dry powder by freeze-concentration using a centrifugal concentrator, and then 100ul of ddH ₂ O was added for redissolution.

(8) The content of CKAP4 in exosomes was detected by Western Blot and CD81 was used as an internal reference. Sample preparation, namely adding a proper amount of exosomes into 2ul of 5× Protein Loading Buffer metal bath at 100 ℃ for 10min, and immediately placing on ice after denaturation. The sample was centrifuged slightly after the temperature was reduced to room temperature.

Loading, namely adding the prepared sample into SDS-PAGE gel, and the electrophoresis program is 70V 35min,120V 90min. The transfer procedure was 300mA 90min.

(9) Blocking with 5% skim milk at room temperature, and after blocking, the corresponding protein bands CKAP4 (63 kDa), CD81 (22-26 kDa) were reduced and incubated overnight with the corresponding antibodies at 4 ℃.

(10) The antibody was recovered and washed three times with 10min each time with TBST. The mouse and rabbit antibodies were incubated at room temperature for 1h. The antibody was recovered and washed three times with 10min each time with TBST.

(11) ECL chemiluminescent solution was applied to the membrane surface and the corresponding bands were detected using a Bio-Rad Berle Chemieoc XRS+ chemiluminescent gel imaging system.

Preparing related solutions:

(1) 5 x electrophoresis buffer: 288g, gly;60.6g, tris;20g, SDS, and finally adding deionized water to fix the volume to 4L. 100mL of 5 Xrunning buffer+400 mL deionized water was used

(2) 14.4G of transfer solution, 3.03g of glycine, 200m of Tris and methanol, and finally adding deionized water to fix the volume to 1L.

(3) 10 XTBS 48.46g, tris 160.21g, naCl, deionized water 1600-1800 mL, pH 7.6+ -0.1 with concentrated hydrochloric acid, and deionized water to 2000mL.

(4) TBST 50mL 10 XTBS+450 mL deionized water+500 ul Tween-20.

SDS-PAGE gel preparation:

10% of separation gel and 5% of concentrated gel. 10% SDS-PAGE protein isolate (5 mL) in a 50mL centrifuge tube, 2.0mL ddH₂O,1.7mL30％Acr-Bis(29:1),1.25mL 1.5M Tris-HCl(pH8.8),0.05mL10％SDS,0.05mL10％APS,0.002mL TEMED, were added in sequence, mixed well into the gel plate and the air bubbles were removed with absolute ethanol. Standing at room temperature, adding 5% SDS-PAGE protein concentrated gel after gel solidification, sequentially sucking 3.4mL ddH₂O,0.85mL 30％Acr-Bis(29:1),0.625mL 1.0M Tris-HCl(pH6.8),0.05mL10％SDS,0.05mL10％APS,0.005mL TEMED,, mixing, adding into gel plate, inserting comb, and solidifying.

Colloidal gold AuNPs characterization of exosome CKAP4

The exosomes of 5637 and SV-HUC-1 cells were observed by Transmission Electron Microscopy (TEM). The aptamer spl3c capable of specific recognition was first attached to colloidal gold particle AuNPs of 5nm in size, and then spl3c-AuNPs was incubated with exosomes at room temperature for 30 minutes. Thereafter, 10. Mu.L of the above exosome sample was dropped on a copper mesh to adsorb for 20 minutes, stained with 2% phosphotungstic acid for 10 minutes, and then washed with 100. Mu.L of pure water 2-3 times to remove the excess dye and unbound AuNPs. Finally, it was dried at room temperature and subjected to TEM imaging (Hitachi H-7000 NAR).

The results are shown in FIG. 2.

Example 3 protein Mass Spectrometry of exosomes

Protein mass spectrometry is carried out on exosomes collected from 5637 and SV-HUC-1 cells respectively, protein abundance scores and fold change of proteins in two groups of exosome samples are compared, and proteins with higher scores and larger differences are selected, namely proteins with obvious differences. As shown in fig. 3, CKAP4 is a protein expressed characteristically in bladder cancer exosomes.

The amino acid sequence of the protein is as follows:

MPSAKQRGSKGGHGAASPSEKGAHPSGGADDVAKKPPPAPQQPPPPPAPHPQQHPQQHPQNQAHGKGGHRGGGGGGGKSSSSSSASAAAAAAAASSSASCSRRLGRALNFLFYLALVAAAAFSGWCVHHVLEEVQQVRRSHQDFSRQREELGQGLQGVEQKVQSLQATFGTFESILRSSQHKQDLTEKAVKQGESEVSRISEVLQKLQNEILKDLSDGIHVVKDARERDFTSLENTVEERLTELTKSINDNIAIFTEVQKRSQKEINDMKAKVASLEESEGNKQDLKALKEAVKEIQTSAKSREWDMEALRSTLQTMESDIYTEVRELVSLKQEQQAFKEAADTERLALQALTEKLLRSEESVSRLPEEIRRLEEELRQLKSDSHGPKEDGGFRHSEAFEALQQKSQGLDSRLQHVEDGVLSMQVASARQTESLESLLSKSQEHEQRLAALQGRLEGLGSSEADQDGLASTVRSLGETQLVLYGDVEELKRSVGELPSTVESLQKVQEQVHTLLSQDQAQAARLPPQDFLDRLSSLDNLKASVSQVEADLKMLRTAVDSLVAYSVKIETNENNLESAKGLLDDLRNDLDRLFVKVEKIHEKV

Example 4 measurement of cell surface mechanical Properties

The cell surface mechanical profile was determined on a JPK Nanowizard 4 atomic force spectrometer with a DNP-10 probe (0.06N/m) and the cells were observed under bright field using an Olympus confocal microscope. Before measuring the Young's modulus of the cells, the spring constant of the cantilever was first calibrated by thermal noise signals, and force curve correcting probe parameters were collected on a cell culture dish filled with ultrapure water. Thereafter, cells seeded in 35mm cell culture dishes were washed 3 times with 1mL of PBS, and finally the cells were placed in RPMI-1640 cell culture medium without FBS. The measurement parameters were set such that the pressing force was 2.0nN, the Z length was 2000nm, the Z scanning speed was 60 μm/s, the scanning area was 60X 60 μm, and the pixel was 256. To fit the experimental data, herz models were selected and the force curves were fitted using JPKSPM data processing software.

Where F is the force generated by the force curve, E is Young's modulus, v is Poisson's ratio (typically 0.2-0.5), R is tip radius, and δ is indentation depth. The results are shown in FIG. 4.

Example 5 cell migration Capacity assay

1) Single cell motion tracking

Cells were seeded in 35mm glass bottom dishes. After cell attachment, cells were washed with PBS and then stained with Hoechst at a concentration of 4. Mu.g/mL for 10 minutes at room temperature. Cells were washed 3 times with PBS and stored in complete medium for use. The change in relative cell position was observed under a 60 x objective, and cell position was recorded every 10 minutes for a total period of 1 hour. Images were analyzed with ImageJ software and each experiment counted more than 50 cells. The results (for exosome effect experiments, the exosome-containing medium and cells were first incubated overnight before cell inoculation, allowing exosomes to enter the cells.) are shown in fig. 5.

2) Scratch test

Cells 5637NC and 5637shCKAP4 were seeded in 96-well plates overnight to allow attachment of the cells, and after attachment of the cells, the original medium was replaced with RPMI-1640 medium without FBS and cultured for 24h. After the cell density reached 80-90%, uniform traces were scored in single layer cells with a streak machine (BioTek) and washed three times to remove suspended cells and cell debris. The scratch healing was then monitored with an inverted microscope for 0, 4, 8 and 12 hours. ImageJ software measured and recorded scratch areas. The results (for exosome effect experiments, exosomes and cells were first incubated overnight before cell inoculation, allowing exosomes to enter cells.) are shown in fig. 5.

3) Transwell experiment

2X 10 ⁴ cells were suspended in 200. Mu.L of complete cell culture medium and seeded in the upper chamber of a 24-well plate (8 μm pore size, corning) overnight to adherence. The next day, the medium in the upper chamber was replaced with fresh cell medium without FBS, and 500 μl of medium containing 20% fetal bovine serum was added to the lower chamber for inducing cell migration. Incubate for 3 days at 37 ℃. Subsequently, the cells were washed 3 times with PBS, and then fixed with 4% paraformaldehyde and 1% crystal violet, respectively, at room temperature for 10min. The non-migrating cells were washed with ddH ₂ O, rubbed with a cotton swab, and images were acquired using BioTek Cytation1CELL IMAGING MultiMode Reader imaging system, imageJ counted for the number of migrating cells. The results (for exosome effect experiments, exosomes and cells were first incubated overnight before cell inoculation, allowing exosomes to enter cells.) are shown in fig. 5.

EXAMPLE 6 liver metastasis of bladder cancer cells in mice

In a tumor model of bladder cancer cells, female thymus-deficient BALB/c (BALB/c-nude mice) mice were purchased from Hunan SJA laboratory animal Co., ltd, and studied using female BALB/c mice of 6 weeks of age. For the exosome-free group, mice were intravenously injected with 200 μl of 5×10 ⁶ 5637shNC and shCKAP of 5637 cells, for the exosome-treated group, shNC and shCKAP of 5637 cells were incubated with or without CKAP4 for 12 hours, then cells were collected to make 200 μl of 5×10 ⁶ cell suspension and injected into mice, after which 5637 of shNC or shCKAP exosomes were intravenously injected every 2 weeks until week 8. Livers were fixed in 4% paraformaldehyde and embedded in paraffin, and bladder cancer cells were analyzed for liver metastasis using hematoxylin-eosin (H & E), ki-67 and CKAP4 staining. The results are shown in FIG. 6.

Example 7 exosomes were collected from urine samples using gradient centrifugation.

The experiment comprises the following specific steps:

(1) The method comprises the steps of collecting morning urine of healthy people (HD), bladder cancer (high grade hBLCA; low grade lBLCA), postoperative (AS), prostate cancer (PRAD), kidney cancer (KIRC), non-urinary tract cancer (nBLCA), benign tumor of the bladder (bBL) patients, transferring urine to a 50mL centrifuge tube at 4000rpm for 30min at 4 ℃, collecting and transferring supernatant to a new 50mL centrifuge tube at 20000rpm for 40min at 4 ℃, and collecting and transferring supernatant to a new 50mL centrifuge tube.

(2) Disposable needle filter 0.22um 33mm filtration and collection of supernatant.

(3) The supernatant was dispensed into Beckman ultracentrifuge tubes (55654), and the low temperature, ultra high speed centrifuge (Beckman_L-100 XP) was programmed at 109000g,1h,4 ℃.

(4) The supernatant was discarded and the pellet was resuspended with pre-chilled DPBS and the low temperature ultra high speed centrifuge program was designed to 110000g,1h,4 ℃.

(5) The supernatant was discarded, the exosomes were resuspended with residual liquid and thoroughly collected with pre-chilled 200-400 ul DPBS.

(6) The collected exosomes were concentrated to a dry powder by freeze-concentration using a centrifugal concentrator, and then 100ul of ddH ₂ O was added for redissolution.

(7) The content of CKAP4 in exosomes was detected by Western Blot and CD81 was used as an internal reference. Sample preparation, namely adding a proper amount of exosomes into a metal bath at 100 ℃ for 10min after 4ul Protein Loading Buffer, and immediately placing the exosomes on ice after denaturation. The sample was centrifuged slightly after the temperature was reduced to room temperature.

(8) Blocking with 5% skim milk at room temperature, and after blocking, the corresponding protein bands CKAP4 (63 kDa), CD81 (22-26 kDa) were reduced and incubated overnight with the corresponding antibodies at 4 ℃.

(9) The antibody was recovered and washed three times with 10min each time with TBST. The mouse and rabbit antibodies were incubated at room temperature for 1h. The antibody was recovered and washed three times with 10min each time with TBST.

(10) ECL chemiluminescent solution covered the membrane surface and the corresponding bands were detected using a Bio-rad bure ChemiDoc xrs+ chemiluminescent gel imaging system.

The results are shown in FIG. 7.

Sequence listing

<110> University of Hunan

<120> Application of CKAP4 reagent in detection specimen and bladder cancer detection kit

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 602

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 1

Met Pro Ser Ala Lys Gln Arg Gly Ser Lys Gly Gly His Gly Ala Ala

1 5 10 15

Ser Pro Ser Glu Lys Gly Ala His Pro Ser Gly Gly Ala Asp Asp Val

20 25 30

Ala Lys Lys Pro Pro Pro Ala Pro Gln Gln Pro Pro Pro Pro Pro Ala

35 40 45

Pro His Pro Gln Gln His Pro Gln Gln His Pro Gln Asn Gln Ala His

50 55 60

Gly Lys Gly Gly His Arg Gly Gly Gly Gly Gly Gly Gly Lys Ser Ser

65 70 75 80

Ser Ser Ser Ser Ala Ser Ala Ala Ala Ala Ala Ala Ala Ala Ser Ser

85 90 95

Ser Ala Ser Cys Ser Arg Arg Leu Gly Arg Ala Leu Asn Phe Leu Phe

100 105 110

Tyr Leu Ala Leu Val Ala Ala Ala Ala Phe Ser Gly Trp Cys Val His

115 120 125

His Val Leu Glu Glu Val Gln Gln Val Arg Arg Ser His Gln Asp Phe

130 135 140

Ser Arg Gln Arg Glu Glu Leu Gly Gln Gly Leu Gln Gly Val Glu Gln

145 150 155 160

Lys Val Gln Ser Leu Gln Ala Thr Phe Gly Thr Phe Glu Ser Ile Leu

165 170 175

Arg Ser Ser Gln His Lys Gln Asp Leu Thr Glu Lys Ala Val Lys Gln

180 185 190

Gly Glu Ser Glu Val Ser Arg Ile Ser Glu Val Leu Gln Lys Leu Gln

195 200 205

Asn Glu Ile Leu Lys Asp Leu Ser Asp Gly Ile His Val Val Lys Asp

210 215 220

Ala Arg Glu Arg Asp Phe Thr Ser Leu Glu Asn Thr Val Glu Glu Arg

225 230 235 240

Leu Thr Glu Leu Thr Lys Ser Ile Asn Asp Asn Ile Ala Ile Phe Thr

245 250 255

Glu Val Gln Lys Arg Ser Gln Lys Glu Ile Asn Asp Met Lys Ala Lys

260 265 270

Val Ala Ser Leu Glu Glu Ser Glu Gly Asn Lys Gln Asp Leu Lys Ala

275 280 285

Leu Lys Glu Ala Val Lys Glu Ile Gln Thr Ser Ala Lys Ser Arg Glu

290 295 300

Trp Asp Met Glu Ala Leu Arg Ser Thr Leu Gln Thr Met Glu Ser Asp

305 310 315 320

Ile Tyr Thr Glu Val Arg Glu Leu Val Ser Leu Lys Gln Glu Gln Gln

325 330 335

Ala Phe Lys Glu Ala Ala Asp Thr Glu Arg Leu Ala Leu Gln Ala Leu

340 345 350

Thr Glu Lys Leu Leu Arg Ser Glu Glu Ser Val Ser Arg Leu Pro Glu

355 360 365

Glu Ile Arg Arg Leu Glu Glu Glu Leu Arg Gln Leu Lys Ser Asp Ser

370 375 380

His Gly Pro Lys Glu Asp Gly Gly Phe Arg His Ser Glu Ala Phe Glu

385 390 395 400

Ala Leu Gln Gln Lys Ser Gln Gly Leu Asp Ser Arg Leu Gln His Val

405 410 415

Glu Asp Gly Val Leu Ser Met Gln Val Ala Ser Ala Arg Gln Thr Glu

420 425 430

Ser Leu Glu Ser Leu Leu Ser Lys Ser Gln Glu His Glu Gln Arg Leu

435 440 445

Ala Ala Leu Gln Gly Arg Leu Glu Gly Leu Gly Ser Ser Glu Ala Asp

450 455 460

Gln Asp Gly Leu Ala Ser Thr Val Arg Ser Leu Gly Glu Thr Gln Leu

465 470 475 480

Val Leu Tyr Gly Asp Val Glu Glu Leu Lys Arg Ser Val Gly Glu Leu

485 490 495

Pro Ser Thr Val Glu Ser Leu Gln Lys Val Gln Glu Gln Val His Thr

500 505 510

Leu Leu Ser Gln Asp Gln Ala Gln Ala Ala Arg Leu Pro Pro Gln Asp

515 520 525

Phe Leu Asp Arg Leu Ser Ser Leu Asp Asn Leu Lys Ala Ser Val Ser

530 535 540

Gln Val Glu Ala Asp Leu Lys Met Leu Arg Thr Ala Val Asp Ser Leu

545 550 555 560

Val Ala Tyr Ser Val Lys Ile Glu Thr Asn Glu Asn Asn Leu Glu Ser

565 570 575

Ala Lys Gly Leu Leu Asp Asp Leu Arg Asn Asp Leu Asp Arg Leu Phe

580 585 590

Val Lys Val Glu Lys Ile His Glu Lys Val

595 600

Claims

1. Use of a reagent for detecting CKAP4 in a specimen in the preparation of a bladder cancer diagnostic preparation, wherein the specimen comprises at least one of tissue, cells, cell exosomes, blood, urine and urine exosomes; the expression level of CKAP4 in the specimen of bladder cancer patients is higher than that in healthy people, patients with urinary system inflammation, or other cancer patients.

2. The use according to claim 1, characterized in that the reagent for detecting CKAP4 in a sample comprises a reagent for detecting the content of CKAP4 protein in a sample.

3. The use according to claim 2, characterized in that the reagent for detecting CKAP4 in a specimen also includes a reagent for extracting CKAP4 protein in the specimen.

4. The use according to claim 1, characterized in that the specimen comprises: at least one of urine and urine exosomes.

5. The use according to claim 4, characterized in that the specimen is urine exosomes.