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Application of urine microvesicle protein as kidney cancer diagnosis marker

A technology of protein markers and vesicles, applied in the field of medical diagnosis, can solve problems such as low survival rate and poor prognosis

Pending Publication Date: 2022-08-02
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

50% of patients are incidentally discovered through imaging examinations during physical examination, and about 16% of renal cell carcinomas have metastasized at the time of diagnosis, and most patients can only be treated with palliative treatment. The prognosis is poor, and the 5-year survival rate is less than 10%

Method used

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  • Application of urine microvesicle protein as kidney cancer diagnosis marker
  • Application of urine microvesicle protein as kidney cancer diagnosis marker
  • Application of urine microvesicle protein as kidney cancer diagnosis marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Construction of lectin-coupled magnetic beads

[0025] 1. Purchase NHS magnetic beads (Solarbio, product number M2450) with a particle size of 2 μm, take 500 μL of magnetic bead suspension into a 1.5 mL EP tube, place the EP tube in a magnetic separation rack, enrich the magnetic beads, and remove the supernatant . Add 1 mL of 4 ℃ pre-cooled Washing Buffer A to a 1.5 mL EP tube, and vortex for 15 s to mix the magnetic beads evenly. Place the EP tube in a magnetic separation rack, enrich the magnetic beads, and remove the supernatant.

[0026] 2. Weigh the lyophilized wheat germ agglutinin powder, and resuspend it into a 3.0 mg / mL protein solution with Coupling Buffer (Solarbio, product number M2450). Add 500 μL of wheat germ agglutinin protein solution to the EP tube in the first step, and vortex for 30 s to mix well. The EP tube was vortexed for 15s, placed on a mixer, and mixed at room temperature for 2h. If the mixing is not uniform, remove the EP tube ...

Embodiment 2

[0030] Example 2 Detection of Urine Microvesicle Protein Spectrum

[0031] 1. Collect urine from patients with renal clear cell carcinoma and control group (including healthy people, benign renal tumors and benign renal cysts), centrifuge at 3000 rpm for 30 minutes at room temperature, remove cell debris, and retain the supernatant.

[0032] 2. Since the surface of extracellular vesicles is rich in glycosylated proteins, these glycosylated proteins can bind to lectins coupled to magnetic beads. Therefore, the prepared lectin-coupled magnetic beads were added to the urine supernatant obtained in the first step, and incubated at room temperature for 1 hour, at which time the extracellular vesicles in the urine would be captured by the magnetic beads.

[0033] 3. Place the EP tube on a magnetic separation rack to magnetically separate and remove the supernatant, and retain the magnetic beads and their captured extracellular vesicles.

[0034] 4. Add 1000 μL of PBS solution (pH 7...

Embodiment 3

[0039] Urine microvesicle protein detection by flow cytometry

[0040] 1. Collect urine from patients with renal clear cell carcinoma and control group (including healthy people, benign renal tumors and benign renal cysts), centrifuge at 3000 rpm for 30 minutes at room temperature, remove cell debris, and retain the supernatant.

[0041] 2. Since the surface of extracellular vesicles is rich in glycosylated proteins, these glycosylated proteins can bind to lectins coupled to magnetic beads. Therefore, 0.5 ml of the prepared lectin-conjugated magnetic beads were added to 1 ml of the urine supernatant obtained in the first step, and incubated at room temperature for 1 hour. At this time, the extracellular vesicles in the urine would be captured by the magnetic beads.

[0042] 3. Place the EP tube on a magnetic separation rack to magnetically separate and remove the supernatant, and retain the magnetic beads and their captured extracellular vesicles. Then, 1000 μL of PBS solutio...

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Abstract

The invention discloses an application of a urine microvesicle protein as a kidney cancer diagnosis marker. The invention relates to a protein marker derived from urine extracellular vesicles related to renal clear cell carcinoma. The protein marker is characterized by being selected from any one or more of the following proteins: CSPG4, AQP1, PLIN2, CA9 and HAVCR1. The invention also discloses application of a reagent for detecting the protein marker in preparation of a reagent for auxiliary diagnosis of renal clear cell carcinoma. The inventor analyzes and finds that five proteins from urine extracellular vesicles can effectively distinguish renal clear cell carcinoma patients from control groups (including healthy people, benign renal tumor patients, benign renal cyst patients and chronic kidney disease patients) in 200 patients through a flow cytometry technology.

Description

technical field [0001] The invention belongs to the field of medical diagnosis, and relates to an application of using urine microvesicle protein as a diagnostic marker for renal cancer. Background technique [0002] Kidney cancer is one of the common malignant tumors of the urinary system. The global incidence continues to rise. About 4% of the malignant disease in adults is renal cancer, which ranks first in the mortality rate of urological tumors every year. Many kidney cancers have no obvious symptoms at an advanced stage, and only about 20% of patients have the three classic symptoms of kidney cancer (hematuria, abdominal mass, and pain). 50% of patients are found incidentally through imaging examinations during physical examination, and about 16% of renal cancers have metastasized at the time of diagnosis. Most patients can only be treated with palliative treatment. The prognosis is poor, and the 5-year survival rate is less than 10%. Therefore, the active search for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886G01N33/574
CPCC12Q1/6886G01N33/57438G01N33/57484C12Q2600/158
Inventor 梁宏伟王嘉凡张衡杨荣
Owner CHINA PHARM UNIV
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