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Application of serum exosome miRNAs marker to early diagnosis of endemic arsenism

An early diagnosis and marker technology, applied in the fields of engineering and clinical medicine, can solve the problem that the application in the early diagnosis and monitoring of prescription arsenic poisoning has not received corresponding attention, achieve easy screening and accurate quantitative analysis, improve Sensitivity and specificity, easily detectable effects

Inactive Publication Date: 2017-02-22
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the application of serum exosomal miRNAs in the early diagnosis and monitoring of endemic arsenicism has not received corresponding attention. The research and development of diagnostic and monitoring kits for corresponding diseases will not only take an international leading position in this field, but also create remarkable economic benefits, and will also be a strong impetus to the prevention and treatment of endemic arsenic poisoning in my country

Method used

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  • Application of serum exosome miRNAs marker to early diagnosis of endemic arsenism
  • Application of serum exosome miRNAs marker to early diagnosis of endemic arsenism
  • Application of serum exosome miRNAs marker to early diagnosis of endemic arsenism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Embodiment 1: Basis for selection and grouping of research objects

[0082] In December 2013, blood samples from early-stage patients with endemic arsenic poisoning and healthy controls that met the requirements were collected from the cooperation unit Guiyang Medical College. After sorting out the sample data, 80 healthy controls and 80 local Patients with early-stage arsenic poisoning were used as experimental subjects for real-time PCR detection of miRNAs expression. Specific sample classification criteria are as follows:

[0083] Group A: Healthy control group (n=80, 20 people for microarray screening, 30 people for phase one verification, and 30 people for independent population verification), without other major systemic diseases.

[0084] Group B: Early stage endemic arsenicosis group (n=80, 20 people for microarray screening, 30 people for first-stage verification, 30 people for independent population verification), clinically diagnosed as early endemic arsenic...

Embodiment 2

[0085] Example 2: Extraction and identification of serum exosomes

[0086] Prepare serum exosome samples: a) Take 250ul serum; b) Add 63ul ExoQuick reagent (4:1), mix gently, incubate at 4 degrees for 30 minutes-1 hour, after incubation, centrifuge at 1500g-10000g for 30 minutes-1 hour , discard the upper waste liquid; c) absorb the supernatant, then centrifuge at 1500g for 5 minutes, carefully absorb the upper liquid, and dissolve the precipitate with 1 / 10 of the original sample volume in deionized water; d) add an appropriate amount of 2.5% pentadiene After the aldehyde was fixed in a 4-degree refrigerator for 2-3 hours, it was sent to the sample detection room for pre-processing of transmission electron microscope detection, and finally identified exosomes, such as figure 1 shown.

Embodiment 3

[0087] Example 3: Extraction of exosome RNA

[0088]Preparation of exosome RNA: a) Add 1ml Trizol to the extracted exosome pellet, mix (repeatedly pipette or vortex), and place at 4°C for 10min; b) Add 200ul chloroform in proportion (1ml Trizol: 200ul chloroform), vigorously Shake and place at 4°C for 15min; c) Centrifuge at 12000g at 4°C for 15min, draw the upper aqueous phase into a new EP tube (350ml); d) Add 500ul of isopropanol in proportion (1ml Trizol: 500ul isopropanol), mix well, 4 Place at ℃ for 10 minutes; e) centrifuge at 12000g at 4℃ for 10min, discard the supernatant, and sink the RNA to the bottom of the tube; f) add 1ml of 75% ethanol according to the proportion (1ml Trizol: 1ml 75% ethanol), shake gently; g) centrifuge at 12000g at 4℃ After 5 minutes, discard the supernatant as much as possible (repeat washing with 75% ethanol once); h) dry at room temperature, add an appropriate amount of DEPC water to dissolve the precipitated RNA; i) store the processed sam...

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Abstract

The invention belongs to the field of gene engineering and clinical medicine and particularly relates to an endemic arsenism early diagnosis associated serum exosome miRNAs marker and application thereof. The marker is a combination of has-miR-155 and has-miR-191. The marker and a primer thereof can be used for preparation of a kit for early diagnosis of endemic arsenism.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and clinical medicine, and specifically relates to serum exosome microRNAs (miRNAs) markers and applications thereof related to the early occurrence of endemic arsenicosis in humans. Background technique [0002] Endemic arsenicosis, referred to as ground arsenic disease, is a biogeochemical disease, which is caused by the loss of skin pigmentation caused by long-term intake of excessive inorganic arsenic through drinking water, air or food by residents living in a specific geographical environment. Or / and excessive sedation, palmoplantar keratosis and cancerous systemic chronic poisoning. Arsenicosis is an endemic disease that seriously endangers human health. In addition to causing skin changes, inorganic arsenic is a human carcinogen identified by the International Center for Cancer Research, which can cause skin cancer, lung cancer, and high incidence of other visceral cancers. I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/113C12N15/11
CPCC12Q1/6883C12Q2600/158C12Q2600/178
Inventor 刘起展罗菲陈超刘欣璐薛均超
Owner NANJING MEDICAL UNIV
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