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Chronic glomerular disease glomerulosclerosis marker and detection kit thereof

A technology for glomerular disease and glomerulosclerosis, applied in the field of biomedicine, can solve the problems of CKD onset hidden, difficult to repeat, unable to monitor, etc., and achieves simple centrifugation, fast detection process, and simple and easy detection method. row effect

Inactive Publication Date: 2018-11-02
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The early onset of CKD is hidden and difficult to find. At present, urinary albumin excretion rate (UAER) is mainly used as the detection index to judge the progress of CKD clinically. However, this index has certain limitations. It is progressing, but the UAER is still within the normal range and cannot be monitored, and sometimes there is a certain deviation between the test results and the actual clinical situation
However, the estimated glomerular filtration rate (eGFR) estimated by detecting creatinine has a certain hysteresis, and the time when the serum creatinine clearance rate decreases is later than the time when the renal disease itself occurs and progresses.
Renal biopsy is the "gold standard" for diagnosing pathological changes in CKD kidneys, but this examination is invasive to a certain extent, and it is difficult to perform it repeatedly clinically

Method used

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  • Chronic glomerular disease glomerulosclerosis marker and detection kit thereof
  • Chronic glomerular disease glomerulosclerosis marker and detection kit thereof
  • Chronic glomerular disease glomerulosclerosis marker and detection kit thereof

Examples

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Embodiment 1

[0041] A kit for chronic glomerular disease glomerulosclerosis markers, including protease inhibitors, annexin V buffer, 0.8μm standard microspheres, 3μm standard microspheres, APC-labeled that can specifically bind to phosphatidylserine Annexin-V flow cytometric fluorescent antibody, PE-labeled podocalyxin protein specifically binds to podocalyxin flow cytometric fluorescent antibody.

Embodiment 2

[0043] Such as figure 1 As shown, the rapid separation of total particles in urine using the kit of the present invention includes the following steps:

[0044] 1. Take 150ml of urine from the subject in the morning to a urine collection bottle, and transfer the urine to a 50ml centrifuge tube at room temperature within 2 hours after collection;

[0045] 2. Centrifuge the urine at 1500g for 10 minutes at room temperature to remove large cells, macromolecular protein complexes and other impurities;

[0046] 3. Transfer the supernatant to another centrifuge tube, add protease inhibitors to every 50ml urine, and store at -80°C. (Protease inhibitor: add 100mmol / L sodium azide 1.67ml, 10mmol / L phenylmethylsulfonyl fluoride 2.5ml and 1mmol / L leupeptin 0.05ml into each 50ml urine)

[0047] 4. Thaw the supernatant and centrifuge at 3000g for 10 minutes, then take the supernatant, and centrifuge again at 18000g for 20 minutes (20°C) to precipitate the corresponding small vesicles to obtain par...

Embodiment 3

[0049] The 0.8μm standard microspheres and 3μm standard microspheres in the kit of Example 1 were used as reference for quantification and positioning during the flow cytometry detection process, and the APC-labeled annexin-V flow cytometry antibody in the kit, and PE-labeled Podocalyxin flow cytometry antibody labels podocyte particles. The specific steps for detecting the absolute count of podocyte particles in urine by flow cytometry are as follows:

[0050] 1. Microparticle immunofluorescence labeling: Take the particle suspension of Example 2 and place it in a 37℃ water bath to quickly melt, take 4 EP tubes, add 100μL of the particle suspension, and add 5μL of PE-podocalyxin antibody and APC-annexin- to the measuring tube. 5μL of V antibody, another tube without antibody was used as a blank control, and the other two single labels were added with 5μL of PE-labeled podocalyxin antibody and 5μL of APC-labeled annexin-V antibody. After incubating on ice at 4°C for 30 minutes i...

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Abstract

The invention discloses a chronic glomerular disease glomerulosclerosis marker and a detection kit thereof. The marker is a sertoli cell particle in urine. The sertoli cell particle has a specific double-membrane-structured extracellular vesicle, a flow cytometry is used to identify the sertoli cell particles in the urine of a chronic glomerular disease patient, further tests are performed to discover the expression difference of the sertoli cell particles in the chronic glomerular disease patient and even in different-degree glomerulosclerosis, the number of the sertoli cell particles in a to-be-detected urine sample is detected, the occurrence and / or development of chronic glomerular disease glomerulosclerosis can be judged or judged in an assistant manner, and the sertoli cell particlein the urine can be used as the marker for the screening or assistant screening of the chronic glomerular disease glomerulosclerosis. The detection kit coordinated with the flow cytometry can effectively detect the release level of the sertoli cell particles in the urine.

Description

Technical field [0001] The invention belongs to the field of biomedical technology, and specifically relates to a chronic glomerular disease glomerulosclerosis marker and a detection kit thereof. Background technique [0002] The gold standard for the diagnosis of kidney disease is “kidney biopsy” to obtain kidney tissue for pathological examination, but this examination is an invasive operation and is difficult to repeat in clinical work. Urine contains a wealth of biological information, including protein, mRNA, etc. The pathophysiological changes of the entire urinary tract system will lead to changes in the composition of urine, so urine is an important window to obtain the biological status of various cells in the urinary system It can also assist in monitoring disease progression and treatment effect. Microparticles are extracellular vesicles with a diameter of 100-1000nm that are produced by cell activation or apoptosis. They are mainly produced by the blebbing form of ce...

Claims

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Application Information

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IPC IPC(8): C12N5/071G01N33/569
CPCC12N5/0686G01N33/56966G01N2800/347
Inventor 马坤岭鲁荐
Owner SOUTHEAST UNIV
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