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Plasma miRNA markers associated with common congenital intestinal aganglionosis and its application

A technology of ganglion cells and markers, applied in the fields of genetic engineering and clinical medicine, can solve problems such as lack of corresponding attention

Active Publication Date: 2016-01-20
NANJING CHILDRENS HOSPITAL AFFILIATED TO NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of miRNA in plasma in the early diagnosis and monitoring of common congenital intestinal aganglionosis has not received corresponding attention. Markers, and the development of diagnostic and monitoring kits for corresponding diseases, will not only create remarkable economic benefits, but will also be a strong impetus to the prevention and treatment of birth defects in my country

Method used

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  • Plasma miRNA markers associated with common congenital intestinal aganglionosis and its application
  • Plasma miRNA markers associated with common congenital intestinal aganglionosis and its application
  • Plasma miRNA markers associated with common congenital intestinal aganglionosis and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0086] Example 1 Research object selection and grouping basis

[0087] From July 2009 to September 2011, the inventor collected common congenital intestinal agangliocytosis children and children with normal non-congenital intestinal agangliocytosis from hospitals such as the Children's Hospital of Nanjing Medical University. Blood and tissue samples (see for clinical diagnostic criteria figure 1 , figure 2 ), after sorting out the sample data, 100 healthy controls (average age: 89.45±9.1 days) and 100 patients with common intestinal aganglionosis (average age: 92.3±7.01 days) were selected. ) As the experimental object of Real-timePCR to detect miRNA expression. The specific sample classification standards are as follows:

[0088] Group A: healthy control group (n=100, 20 people are screened by chip, 40 people are first-phase verification, 40 people are independent population verification):

[0089] 1. Age between 0 and 6 months;

[0090] 2. No digestive system diseases;

[0091] 3....

Embodiment 2

[0100] Example 2 Pathological Diagnosis Classification of Research Object

[0101] Anorectal manometry, imaging examination, and histological examination of the rectal wall are used to diagnose intestinal agangliosis and preliminarily determine the type of children with past delayed meconium discharge, repeated defecation difficulties, abdominal distension, and low intestinal obstruction. Anorectal manometry is mainly manifested as lack of internal anal sphincter relaxation reflex; anal canal rhythmic contraction is significantly reduced; resting pressure of internal rectal sphincter is higher than normal. The imaging findings are as follows: 1. There is an obvious transitional separation area between the lesion and the expansion, which is a "cone" shape; 2. The lesion starts from the anus and extends upward to the distal end of the sigmoid colon; 3. The lesion is innervated abnormally Therefore, irregular shrinkage can be seen; 4. Barium retention, which may not be discharged af...

Embodiment 3

[0102] Example 3 TaqmanmiRNAarray screening

[0103] Prepare cDNA samples: a) Take 100μl plasma; b) Add 900μl Trizol, shake and mix, 4℃, 12000 rpm for 15 minutes, discard the lower layer of waste; c) Add 1.5 times the volume of the supernatant absolute ethanol, shake and mix, turn To the spin column, centrifuge at 12,000 rpm for 15 seconds, and discard the lower layer of waste; d) Add 700μl RWT buffer to the spin column, centrifuge at 10,000 rpm for 15 seconds, and discard the lower layer of waste. e) Add 500μl RPE buffer to the spin column, centrifuge at 10000rpm for 15 seconds, and discard the lower layer. f) Repeat e. g) Add the spin column to a new 2ml tube and centrifuge at 10000rpm for 1 minute to remove the RPE buffer. h) Add 50μl DEPC-treated water to the column and centrifuge at 12000rpm to collect RNA. i) Then cDNA is obtained by RNA reverse transcription reaction. The reverse transcription reaction system includes 4μl of 5×AMV buffer, 2μl of 10mM dNTP mixture (Taka...

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Abstract

The invention belongs to the fields of genetic engineering and clinical medicines, and discloses a plasma miRNA (micro ribonucleic acid) marker associated with common type congenital intestinal canal total colonic aganglionosis and an application thereof. The marker is selected from more of hsa-miR-31, hsa-miR-147 and hsa-miR-206. The marker has specificity and sensitivity to the common type congenital intestinal canal total colonic aganglionosis, can be used for the preparation of reagents for diagnosing or monitoring the common type congenital intestinal canal total colonic aganglionosis, can avoid invasive diagnosis, can be used for screening and diagnosis in an early stage, and can be used for detecting repeatedly and is easy for dynamic monitoring.

Description

Invention field [0001] The invention belongs to the fields of genetic engineering and clinical medicine, and relates to plasma microribonucleic acid (microRNA, miRNA) markers and applications thereof related to the occurrence of common congenital intestinal aganglionosis. Background technique [0002] Common congenital anorectal malformation (NCA) is a common digestive tract malformation in pediatric surgery, second only to congenital anorectal malformations in the occurrence of congenital digestive tract malformations. CA is characterized by the absence of ganglion cells in the distal gastrointestinal tract, which is mainly manifested as the migration of enteric nerves during the development of the enteric nervous system or abnormal development after migration, resulting in the absence of ganglion cells in the distal intestinal tract, and the function of the distal bowel is affected. However, the main clinical symptoms are difficulty in defecation, abdominal distension and intes...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/11C12Q1/68
Inventor 唐维兵唐俊伟陆春城吴炜秦晶晶李波周志刚刘康
Owner NANJING CHILDRENS HOSPITAL AFFILIATED TO NANJING MEDICAL UNIV
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