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Application of seminal plasma extracellular vesicle HIST1H2BA protein

A protein and supporting cell technology, applied in the field of protein detection, can solve the problems of lack of spermatogenic cells and inability to obtain sperm

Active Publication Date: 2021-04-13
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported that the successful extraction of sperm is closely related to the histopathology of the testis. There are spermatogenic cells in the testis of HS and MA patients, so there is a high probability that sperm can be obtained. unable to obtain sperm

Method used

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  • Application of seminal plasma extracellular vesicle HIST1H2BA protein
  • Application of seminal plasma extracellular vesicle HIST1H2BA protein
  • Application of seminal plasma extracellular vesicle HIST1H2BA protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1. Application of PRM relative quantitative technology to verify testis-specific sEV protein in three pathological types of NS, NOA and OA patients

[0019] 1. Experimental materials:

[0020] Semen analysis showed a normal sperm count (≥15.0 × 10 6 / mL), normal pH (7.20-8.00), normal sperm motility (32.0-100%) are NS. NOA inclusion criteria: 1) Three centrifugation at 3000rpm for 15 minutes without detection of sperm; 2) Testicular histopathology showed reduced spermatogenesis or no germ cells; Exclusion criteria: 1) Known acquired diseases, such as chemotherapy, double Lateral cryptorchidism, testicular torsion; 2) Genetic factors, such as karyotype aberration, y chromosome AZF deletion. The pathological type of NOA was determined by the pathological analysis of testicular tissue obtained by multi-point needle biopsy by hematoxylin and eosin (H&E) staining. Testicular aspiration or percutaneous epididymis aspiration (PESA) to obtain a large number of sperm ...

Embodiment 2

[0028] Example 2. Western blot reveals the expression of HST1H2BA in three pathological types of NS, NOA and sEV of OA patients

[0029] 1. Experimental materials:

[0030] sEV protein in Example 1.

[0031] 2. Experimental process:

[0032] (1) Add 200 μL protein lysis buffer (8 M Urea, 75 mM NaCl, 50 mM Tris, pH8.2, 1% vol / vol EDTA-free protease inhibitor, 1 mM NaF, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate to the sEV sample , 10 mM sodium pyrophosphate), vortexed for 1 hour after sonication, and centrifuged at 40,000 g for 1 hour, all at 4°C.

[0033] (2) Each 10 μg protein sample was separated by SDS / PAGE gel and transferred to a polyvinylidene fluoride membrane. After milk was blocked at room temperature for 2 hours, primary antibodies were used: rabbit anti-HIST1H2BA (1:1000, ab178426, Abcam), rabbit Anti-ALIX (1:1000, 12422-1-AP, Proteintech) was incubated overnight.

[0034](3) After washing the membrane, use the secondary antibody: goat anti-rabbit IgG (1...

Embodiment 3

[0036] Example 3. Localization of HIST1H2BA in human testis

[0037] To better understand the role of testis-specific HIST1H2BA in NOA typing diagnosis, we further investigated its expression and localization in human testis.

[0038] 1. Experimental materials

[0039] Testicular samples from patients with testicular biopsy including euspermatogenic obstructive azoospermia-OA, hypospermatogenicity-HS, maturation arrest-MA, and Sertoli cell-only syndrome-SCO.

[0040] 2. Experimental process

[0041] (1) Fix testicular biopsy tissue with modified Davidson's liquid fixative for 48 hours. After that, the tissue was dehydrated with increasing concentrations of ethanol (70%, 80%, 90%, 100%), and the ethanol was replaced by xylene, embedded in paraffin, and serially sectioned into 5 μm thick sections.

[0042] (2) Place the slices in 3% methanol diluted hydrogen peroxide and incubate at 37°C for 10 minutes to remove endogenous peroxidase. After antigen retrieval, block with 1% BS...

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Abstract

The invention discloses application of seminal plasma extracellular vesicle HIST1H2BA protein, belongs to the field of protein detection, and particularly relates to application of the seminal plasma extracellular vesicle HIST1H2BA protein in biomarkers of seminal plasma extracellular vesicles of different pathological types of non-obstructive azoospermia. The HIST1H2BA provided by the invention can be used for distinguishing NOA-SCO from other pathological types and predicting the spermatogenesis condition in testis.

Description

technical field [0001] The invention relates to the field of protein detection, in particular to the application of biomarkers of seminal plasma extracellular vesicles of different pathological types of non-obstructive azoospermia. Background technique [0002] Male infertility accounts for about half of all infertility cases and affects one in six couples worldwide. A considerable proportion of male infertility is accompanied by azoospermia. Clinically, it is often divided into obstructive azoospermia (OA) with normal testicular spermatogenic function but sperm transport obstacles caused by reproductive tract obstruction, and non-functional azoospermia (OA) with testicular failure itself. There are two types of obstructive azoospermia (NOA). The most common manifestation of azoospermia is NOA, accounting for approximately 60% of all azoospermia cases. According to the histopathological examination of the testis, NOA can be divided into three main pathological types: low...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/689G01N2333/47G01N2800/367
Inventor 郭雪江沙家豪杨晓玉郭曰帅姚礼萍
Owner NANJING MEDICAL UNIV
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