Tumor serum exosome specific molecular marker for renal clear cell carcinoma and kit and detection reagent thereof

Abstract

The invention discloses a tumor serum exosome specific molecular marker of renal clear cell carcinoma as well as a kit and a detection reagent of the tumor serum exosome specific molecular marker. The specific molecular marker is CD147, and the specific molecular marker also comprises CD63. The expression of the CD147 in tumor cells of the renal clear cell carcinoma is obviously increased, and the content of the CD147 on serum exosomes of patients with the renal clear cell carcinoma is obviously higher than that of healthy people, bladder cancer patients and prostate cancer patients. Therefore, the compound can be used as a specific biomarker of the renal clear cell carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of molecular markers, and in particular to a tumor serum exosome-specific molecular marker for renal clear cell carcinoma and a kit and a detection reagent thereof. Background Art

[0002] Exosomes are a type of nano-scale vesicle structure secreted by cells, with a size of about 30 to 100 nm, which are distributed in a variety of body fluids such as blood, saliva, and urine. They contain a variety of proteins, lipids, messenger RNA (mRNA), and microRNA (microRNA), etc., which are biologically active and can be absorbed by receptor cells to achieve intercellular material transport and information transmission. They play a key role in the occurrence and development of cancer. At the same time, these molecules can reflect the physiological and pathological states of their parent cells. For example, exosomes released by tumor cells contain specific tumor markers. Secondly, biological molecules such as RNA and proteins in exosomes may form stable complexes with some proteins, sugar groups, etc. on the exosome membrane, which also helps to maintain the stability of these molecules. Therefore, exosomes can be regarded as ideal biomarkers for non-invasive early cancer diagnosis and efficacy evaluation for in vitro diagnosis, but how to achieve rapid and efficient analysis of exosomes in complex biological sample backgrounds (such as plasma, urine, etc.) is still challenging. The tetraspanin family, such as CD63, CD9, and CD81, has been shown to be useful as exosome markers for detecting exosomes, but they are not tumor-specific and cannot distinguish between exosomes produced by cancer cells and healthy cells, nor can they identify which specific tumor cells produce the exosomes.

[0003] In recent years, it has been found that tumor-derived EVs (T-EVs) also express a variety of tumor surface markers, such as epithelial cell adhesion molecule (Epcam), epidermal growth factor receptor (EGFR), and CA125. Using these biomarkers to track EVs / T-EVs in body fluids has become a non-invasive cancer liquid biopsy method with broad potential application prospects, but it is still challenging to identify and quantify EVs in clinical samples. At present, there are many methods for separating exosomes, and ultracentrifugation is widely regarded as the "gold standard" for exosome separation. This method relies on the size and density differences between exosomes and other components for separation, but it has a big disadvantage. It is a multi-step, labor-intensive and expensive process, and it will destroy the integrity of exosomes. The exosome capture technology developed in recent years is closely related to immune affinity and is often used to produce high-purity exosomes. Magnetic beads are a material that can be modified to bind to target proteins on the membrane surface and play a core role in capture technology. Exosomes contain a variety of membrane proteins on their surface, which can be enriched using antibody-coated magnetic beads. However, the high cost and low yield of magnetic bead-based separation strategies limit their further development and application. At the same time, some detection technologies that can analyze and quantify exosomes have been developed for research and clinical applications. The most commonly used are nanoparticle tracking analysis (NTA) and nano-flow cytometry (NFCM). However, the separation and detection of these two technologies are performed separately, which increases the complexity of the operation, time consumption and cost. Therefore, a faster, more sensitive and low-cost method for capturing and detecting exosomes is needed. Summary of the invention

[0004] The purpose of the present invention is to provide a tumor serum exosome-specific molecular marker for renal clear cell carcinoma - CD147 in extracellular exosomes.

[0005] To achieve the above object, the present invention provides a tumor serum exosome-specific molecular marker for renal clear cell carcinoma, characterized in that the specific molecular marker is CD147.

[0006] Furthermore, CD63 is also included.

[0007] The present invention also provides a kit, characterized in that it comprises the specific molecular marker.

[0008] The present invention also provides an anti-CD147 plasmon resonance biosensor for detecting the specific molecular marker according to claim 1 or 2, characterized in that the preparation method of the anti-CD147 plasmon resonance biosensor is:

[0009] S1. Preparation of plasmon metasurface sensor:

[0010] (1) Nanoimprinting: First, the nanopore array was imprinted on the PDMS flexible substrate through a 4-inch silicon nanopore array template at a pressure of 30±3 bar. Then, a 4-inch silicon wafer with a polymer resist spin-coated to a thickness of 250±5 nm was covered on the PDMS nanopore array and imprinted under ultraviolet light for 60±3 seconds.

[0011] (2) Plasma etching: Use a deep silicon etching system to perform oxygen plasma etching on the silicon wafer to remove the residual UV resist from nanoimprinting. The etching time is 7±2 seconds and the oxygen flow rate is 100±10sccm. Then, use SF6 and C4F8 to etch the silicon wafer for 35±3 seconds with a gas flow rate of 50±3sccm, followed by oxygen plasma etching for 100±3 seconds to remove the resist.

[0012] (3) Electron beam evaporation: a chromium adhesion layer and a gold layer are sequentially deposited on a silicon wafer by magnetron sputtering, wherein the chromium adhesion layer is deposited for 17±1 seconds, the power is 200±5 W, and the argon gas flow rate is 60±3 sccm, and the gold layer is deposited for 100±10 seconds, the power is 400±20 W, and the argon gas flow rate is 60±3 sccm, to obtain a 4-inch metasurface wafer;

[0013] (4) Cut the 4-inch metasurface disc into 3×3 mm 2 The sensor chip is used to obtain a plasmon metasurface sensor;

[0014] S2. Activating the plasmon metasurface: The plasmon metasurface sensor is placed in a 12 mmol / L MUA ethanol solution to form a MUA self-assembled layer; preferably, stored for 6-10 hours;

[0015] S3. Activation of MUA: After washing with deionized water, the mixture was immersed in a mixture of 400mmol / L EDC solution and 100mmol / L NHS solution to activate MUA; preferably, the mixture was immersed for 0.5-1 hour;

[0016] S4. Antibody coupling: The plasmon metasurface chip obtained in S3 is immersed in a 40 μg / mL anti-CD147 solution for incubation, so that the anti-CD147 in the anti-CD147 solution is combined with the activated MUA on the plasmon metasurface to functionalize the chip; preferably, the immersion time is 0.5-1 hour;

[0017] S5. Blocking MUA that is not linked to antibodies: Continue to add 1±3% BSA solution to block MUA that is not linked to anti-CD147.

[0018] The expression of CD147 in renal clear cell carcinoma tumor cells is significantly increased, and the content of CD147 in serum exosomes of renal clear cell carcinoma patients is significantly higher than that of healthy people, bladder cancer patients, and prostate cancer patients. Compared with the detection of CD63, which can only distinguish between tumor patients and healthy people, the detection of CD147 can also distinguish between clear cell carcinoma and other cancers, as well as healthy people, so it can be used as a specific biomarker for renal clear cell carcinoma.

[0019] The present invention is a biomarker detection method for detecting serum exosome membrane protein CD147 to distinguish renal clear cell carcinoma and healthy patients using a highly sensitive label-free exosome detection method. This exosome detection method has the advantages of fast detection speed, high accuracy, simple operation, and low detection cost. Compared with CD63, the use of CD147 as a biomarker can specifically distinguish renal clear cell carcinoma, healthy people and other tumor patients.

[0020] CD147 is a glycoprotein widely present in various tissues and organs of the human body. It belongs to the immunoglobulin superfamily and participates in many physiological processes of the body. It has been proven that its expression increases in a variety of tumor cells in vivo and in vitro, promoting tumor invasion and metastasis. For example, studies have found that the expression of CD147 in primary breast cancer tissue is related to tumor size and stage, and can predict poor prognosis. Currently, monoclonal antibodies targeting CD147 are used in clinical practice. For example, Habl8 is a therapeutic monoclonal antibody for liver cancer, and anti-CD147 monoclonal antibodies also have a significant inhibitory effect on tumor growth in the treatment of head and neck squamous cell carcinoma. However, there is currently no relevant research revealing that CD147 present in extracellular exosomes can be used as a tumor-specific molecular marker for renal clear cell carcinoma.

[0021] The present invention uses anti-CD147 plasmon resonance biosensor (CD147-LPM) for non-destructive sensing detection of exosomes, which can achieve high-sensitivity detection as low as 300 / mL exosome solution. The detection method has high accuracy and is simple to operate. The experimental comparison between the patient and healthy groups was performed using a two-sample t-test, P<0.001. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The expression of CD147 protein in exosomes of renal clear cell carcinoma cell fluid is higher than that in exosomes of renal tubular epithelial cell fluid.

[0023] Figure 2 This is an immunohistochemical analysis of the cancer tissue and adjacent normal tissue of a patient with renal clear cell carcinoma.

[0024] Figure 3 is a scanning electron microscope image of the top view of the anti-CD147 plasmon resonance biosensor (CD147-LPM).

[0025] Figure 4 This is a scanning electron microscope image of the anti-CD147 plasmon resonance biosensor that captured exosomes in a renal cancer serum sample. The red circles indicate the exosomes captured by the sensor.

[0026] Figure 5 The exosomes were purified by ultra-high-speed centrifugation separation from serum, and the CD147 expression profiles of healthy people (H), bladder cancer (BC), prostate cancer (PC) and clear cell renal carcinoma (ccRCC) were analyzed based on nano-flow cytometry (NFCM) and anti-CD147 plasmon resonance biosensor (CD147-LPM).

[0027] Figure 6 This is a graph showing the results of an experiment using an anti-CD147 plasmon resonance biosensor (CD147-LPM) to detect different concentrations of renal cancer exosomes.

[0028] Figure 7 The anti-CD147 plasmon resonance biosensor (CD147-LPM) was used to directly detect serum samples, H (n=5), BC (n=6), PC (n=6) and ccRCC (n=6). The results showed that CD147 could distinguish ccRCC patients from the above four groups of people, but CD63 could not. DETAILED DESCRIPTION

[0029] Embodiments of the present invention are described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by reference to the accompanying drawings are exemplary, are intended to be used to explain the present invention, and are not to be construed as limitations of the present invention. Those who do not indicate specific techniques or conditions in the embodiments are carried out according to the techniques or conditions described in the documents in this area or according to the product specification. Those who do not indicate the manufacturer of reagents or instruments used are all conventional products that can be obtained commercially.

[0030] Embodiment 1:

[0031] 1. Ultra-high-speed centrifugation was used to collect and purify exosomes from the culture supernatant of renal clear cell carcinoma cells and renal tubular epithelial cells, and proteins were extracted for immunoblotting (WB) to analyze the expression of CD147, CD63, and CD81. The results are shown in Figure 1 , Figure 1 The expression of CD147 protein in exosomes of renal clear cell carcinoma cell fluid is higher than that in exosomes of renal tubular epithelial cell fluid. Figure 1CD63 and CD81 are commonly used identification markers of exosomes, which are used to indicate that the extracted proteins are indeed exosome proteins; GAPDH is the experimental internal reference protein; HK-2 is renal tubular epithelial cells; A498, 786-0, and ACHN are renal clear cell carcinoma cell lines. Figure 1 It can be seen that the expression of CD147 protein in renal clear cell carcinoma cell fluid exosomes is higher than that in renal tubular epithelial cells.

[0032] 2. Immunohistochemistry was used to analyze the protein expression level of CD147 in renal clear cell carcinoma tissue and adjacent normal tissue. The results showed that the expression of CD147 in cancer tissue was significantly higher than that in adjacent normal tissue. Figure 2 .

[0033] 3. The exosomes were separated and purified by ultra-high-speed centrifugation from the serum of healthy people (H), bladder cancer (BC), prostate cancer (PC) and clear cell renal carcinoma (ccRCC) patients, and the CD147 expression profile was analyzed based on nano-flow cytometry (NFCM) and anti-CD147 plasmon resonance biosensor (CD147-LPM), respectively. The results of exosome detection by CD147-LMB showed good consistency with the detection results of traditional nano-flow cytometry (NFCM). The results are shown in Figure 5 .

[0034] 4. Taking advantage of the specificity of CD147, CD63-LMB and CD147-LMB were applied to more serum samples from healthy people (H), bladder cancer (BC), prostate cancer (PC), and clear cell renal carcinoma (ccRCC) individuals. The results showed that CD63-LMB detection can effectively distinguish cancer samples from healthy samples, but the statistical distinction for BC, PC and ccRCC was not significant. However, the detection statistics of CD147-LMB showed its excellent performance in distinguishing ccRCC from the other two urological tumors. Combining serum tests of CD63-LMB and CD147-LMB can distinguish ccRCC from healthy individuals and other patients with urological tumors. Results are shown in Figure 7 .

[0035] The above experiments verified that: CD147 expression in renal clear cell carcinoma tumor cells increased significantly, and the content of CD147 in serum exosomes of renal clear cell carcinoma patients was significantly higher than that of healthy people, bladder cancer patients, and prostate cancer patients. Compared with the detection of CD63, which can only distinguish between tumor patients and healthy people, the detection of CD147 can also distinguish between clear cell carcinoma and other cancers, as well as healthy people, so it can be used as a specific biomarker for renal clear cell carcinoma.

[0036] Example 2: Preparation of anti-CD147 plasmon resonance biosensor:

[0037] A. Experimental materials: polydimethylsiloxane (PDMS), 4-inch silicon wafer, 11-mercaptoundecanoic acid (MUA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), anhydrous ethanol, phosphate buffered saline (PBS), bovine serum albumin (BSA), anti-CD147 antibody;

[0038] B. Experimental instruments: deep silicon etching system, magnetron sputtering coating machine;

[0039] C. Steps:

[0040] S1. Preparation of plasmon metasurface sensor:

[0041] (1) Nanoimprinting: First, the nanopore array was imprinted on the PDMS flexible substrate through a 4-inch silicon nanopore array template under a pressure of 30 bar. Then, a 4-inch silicon wafer with a polymer resist spin-coated to a thickness of 250 nm was covered on the PDMS nanopore array and imprinted under ultraviolet light for 60 seconds.

[0042] (2) Plasma etching: The silicon wafer was etched with oxygen plasma using a deep silicon etching system to remove the residual UV resist from nanoimprinting. The etching time was 7 seconds and the oxygen flow rate was 100 sccm. After that, the silicon wafer was etched with SF6 and C4F8 for 35 seconds at a gas flow rate of 50 sccm, followed by oxygen plasma etching for 100 seconds to remove the resist.

[0043] (3) Electron beam evaporation: A chromium adhesion layer and a gold layer were sequentially deposited on the silicon wafer by magnetron sputtering, wherein the chromium adhesion layer was deposited for 17 seconds, the power was 200 W, and the argon gas flow rate was 60 sccm, and the gold layer was deposited for 100 seconds, the power was 400 W, and the argon gas flow rate was 60 sccm. A 4-inch metasurface wafer was obtained.

[0044] (4) Cut the 4-inch metasurface disc into 3×3 mm 2 The sensor chip is used to obtain a plasmon metasurface sensor.

[0045] S2. Activating the plasmon metasurface: preparing a 12 mmol / L MUA (11-mercaptoundecanoic acid solution) ethanol solution, and placing the plasmon metasurface sensor prepared by the above method in the MUA ethanol solution for 8 hours to form a MUA self-assembled layer;

[0046] S3. Activate MUA: prepare a mixed solution of 400mmol / L EDC (ethyldimethylaminopropylcarbodiimide solution) and 100mmol / L NHS (N-hydroxysuccinimide), first clean the plasmon metasurface chip in S2 with deionized water, and then soak it in a mixture of 200μl 400mmol / L EDC solution and 100mmol / L NHS solution for 0.5 to 1.0 hour to activate MUA;

[0047] S4. Antibody coupling: The plasmon metasurface chip obtained in S3 is immersed in a 40ug / mL anti-CD147 solution (i.e., anti-CD147 PBS solution) for 0.5 hours for incubation, so that the anti-CD147 in the anti-CD147 solution is combined with the activated MUA on the plasmon metasurface to functionalize the chip;

[0048] S5. Blocking the MUA not linked to the antibody: Continue to add 200 μl of 1% BSA (bovine serum albumin) solution to block the MUA not linked to anti-CD147. The anti-CD147 plasmon resonance biosensor (CD147-LMB) is obtained. The results are shown in Figure 3 , Figure 3 Top view image of the sensor under a scanning electron microscope.

[0049] Similarly, the preparation method of the anti-CD63 plasmon resonance biosensor is the same, except that anti-CD147 is replaced by anti-CD63.

[0050] S6. Add 2 μl of serum to the anti-CD147 plasmon resonance biosensor for exosome determination. Scanning electron microscopy shows the captured exosomes on the sensor. Figure 4 The red circles indicate the exosomes captured by the sensor.

[0051] Example 3: Effect verification test

[0052] Sample source: Serum from patients with renal cancer.

[0053] Sample pretreatment: 1. Use ordinary vacuum blood collection tubes to collect peripheral blood (arterial or venous blood) samples from subjects, without EDTA or heparin, draw 3 ml, let it stand at room temperature for 30 minutes, and wait for blood coagulation to be complete;

[0054] 2. Serum was obtained by centrifugation at room temperature, 3000 rpm, 15 min;

[0055] 3. Purify serum exosomes by ultra-high-speed centrifugation, 4°C, 100,000 rpm, 70 min;

[0056] 4. Dilute the exosomes with PBS and use NCFM to calculate the original solution concentration to be 6*10 10 / ml, dilute this original solution to obtain a concentration of 3*10 11 / mL, 3*10 10 / mL, 3*10 9 / mL, 3*10 8 / mL, 2*10 8 Pieces / mL, 1*10 8 / mL, 3*10 7 Pieces / mL, 1*10 7 / mL, 3*10 6 / mL, 3*10 5 / mL, 3*10 4 / mL, 3*10 3 / mL, 3*10 2 Pieces / mL of test solution;

[0057] 5. Detection: The test solution was added to the anti-CD147 plasmon resonance biosensor to determine the exosome content. The relationship between the exosome content and the offset was determined using the four-combination equation y = 0.94-0.88 / [1+(x / 2.23×10 6 ) 0.56 ] for fitting, where the x-axis is the exosome content (unit: pieces / mL) and the y-axis is the offset (unit: nm). The experiment was calculated by NCFM. Figure 6 .

[0058] Offset determination: Biomarker molecules bound to the sensor surface will cause changes in the environmental refractive index and thus change the resonance wavelength, thereby achieving quantitative and real-time detection of biomarkers. The biomarker exosome membrane protein CD147 is detected by anti-CD147 plasmon resonance biosensor, and the offset is measured using optical offset. The above-mentioned four-in-one equation is then used to calculate the content of exosome CD147.

[0059] Analysis results: When the exosomes are serum exosomes from patients with renal cancer and the unit of measurement of exosomes is pieces / mL, the anti-CD147 plasmon resonance biosensor tested the concentration of 3*10 11 / mL, 3*10 10 / mL, 3*10 9 / mL, 3*10 8 / mL, 2*10 8 Pieces / mL, 1*10 8 / mL, 3*10 7Pieces / mL, 1*10 7 / mL, 3*10 6 / mL, 3*10 5 / mL, 3*10 4 / mL, 3*10 3 / mL, 3*10 2 / mL test solution, producing 6.94nm, 6.72nm, 6.74nm, 6.56nm, 5.21nm, 4.43nm, 3.29nm, 2.16nm, 0.96nm, 0.93nm, 0.74nm, 0.58nm, 0.37nm offsets respectively. Combined with the above formula, from the numerical point of view, when the exosome concentration is higher, the offset is also greater, and the CD147 expression content is higher.

[0060] CD147 is also expressed in normal human serum, but at a low level. When the content is higher than 4.42nm, it is a tumor sample. In this embodiment, the deviation of CD147 in the 6 ccRCC patients detected in Example 1 was greater than 6nm, and that in the 4 healthy patients was less than 2.8nm.

[0061] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A tumor serum exosome-specific molecular marker for renal clear cell carcinoma, characterized in that: The specific molecular marker is CD147.

2. The specific molecular marker according to claim 1, characterized in that: Also included is CD63.

3. A kit, characterized in that: Comprising the specific molecular marker according to claim 1 or 2.

4. An anti-CD147 plasmon resonance biosensor for detecting the specific molecular marker according to claim 1 or 2, characterized in that: The preparation method of the anti-CD147 plasmon resonance biosensor is as follows: S1. Preparation of plasmon metasurface sensor: (1) Nanoimprinting: First, the nanopore array was imprinted on the PDMS flexible substrate through a 4-inch silicon nanopore array template at a pressure of 30±3 bar. Then, a 4-inch silicon wafer with a polymer resist spin-coated to a thickness of 250±5 nm was covered on the PDMS nanopore array and imprinted under ultraviolet light for 60±3 seconds. (2) Plasma etching: Use a deep silicon etching system to perform oxygen plasma etching on the silicon wafer to remove the residual UV resist from nanoimprinting. The etching time is 7±2 seconds and the oxygen flow rate is 100±10sccm. Then, use SF6 and C4F8 to etch the silicon wafer for 35±3 seconds with a gas flow rate of 50±3sccm, followed by oxygen plasma etching for 100±3 seconds to remove the resist. (3) Electron beam evaporation: a chromium adhesion layer and a gold layer are sequentially deposited on a silicon wafer by magnetron sputtering, wherein the chromium adhesion layer is deposited for 17±1 seconds, the power is 200±5 W, and the argon gas flow rate is 60±3 sccm, and the gold layer is deposited for 100±10 seconds, the power is 400±20 W, and the argon gas flow rate is 60±3 sccm, to obtain a 4-inch metasurface wafer; (4) Cut the 4-inch metasurface disc into 3×3 mm 2 The sensor chip is used to obtain a plasmon metasurface sensor; S2. Activating the plasmon metasurface: The plasmon metasurface sensor is placed in a 12 mmol / L MUA ethanol solution to form a MUA self-assembled layer; preferably, stored for 6-10 hours; S3. Activation of MUA: After washing with deionized water, the mixture was immersed in a mixture of 400mmol / L EDC solution and 100mmol / L NHS solution to activate MUA; preferably, the mixture was immersed for 0.5-1 hour; S4. Antibody coupling: The plasmon metasurface chip obtained in S3 is immersed in a 40 μg / mL anti-CD147 solution for incubation, so that the anti-CD147 in the anti-CD147 solution is combined with the activated MUA on the plasmon metasurface to functionalize the chip; preferably, the immersion time is 0.5-1 hour; S5. Blocking MUA that is not linked to antibodies: Continue to add 1±3% BSA solution to block MUA that is not linked to anti-CD147.

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