Plasma miRNA (micro ribonucleic acid) marker associated with common type congenital intestinal canal total colonic aganglionosis and application thereof

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: 2014-05-21
NANJING CHILDRENS HOSPITAL AFFILIATED TO 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 miRNA in plasma in the early diagnosis and monitoring of common congenital intestinal aganglionosis has not received corresponding attention. Markers, and the development of di

Method used

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  • Plasma miRNA (micro ribonucleic acid) marker associated with common type congenital intestinal canal total colonic aganglionosis and application thereof
  • Plasma miRNA (micro ribonucleic acid) marker associated with common type congenital intestinal canal total colonic aganglionosis and application thereof
  • Plasma miRNA (micro ribonucleic acid) marker associated with common type congenital intestinal canal total colonic aganglionosis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Embodiment 1 Research Object Selection and Grouping Basis

[0087] From July 2009 to September 2011, the inventor collected children with common congenital intestinal aganglionosis and normal non-congenital intestinal aganglionosis from the Children's Hospital Affiliated to Nanjing Medical University and other hospitals that met the requirements. Blood and tissue samples (for clinical diagnostic criteria see figure 1 , figure 2), from which 100 healthy controls (average age: 89.45±9.1 days) and 100 patients with intestinal common congenital intestinal aganglionosis (average age: 92.3±7.01 days) were selected to meet the requirements ) as the experimental object of Real-time PCR detection of miRNA expression. Specific sample classification criteria are as follows:

[0088] Group A: healthy control group (n=100, 20 people for microarray screening, 40 people for first-stage verification, 40 people for independent crowd verification):

[0089] 1. Age between 0 and 6 mo...

Embodiment 2

[0100] Embodiment 2 research object pathological diagnosis type

[0101] Anorectal manometry, imaging examination, and rectal wall histological examination were used to diagnose intestinal aganglionosis and preliminarily clarify its type for children with delayed meconium discharge, repeated defecation difficulties, abdominal distension, and low intestinal obstruction in the past. Anorectal manometry mainly shows the absence of internal anal sphincter relaxation reflex; the rhythmic contraction of anal canal is significantly reduced; the resting pressure of internal rectal sphincter is higher than normal. Imaging examination showed that: 1. There was an obvious transition zone between the lesion segment and the dilation segment, showing a "cone" shape; 2. The lesion segment extended upward from the anus to the distal end of the sigmoid colon; 3. The innervation of the lesion segment was abnormal Therefore, it can be seen that there are irregular contractions; 4. The retention ...

Embodiment 3

[0102] Example 3 Taqman miRNA array screening

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

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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

field of invention [0001] The invention belongs to the fields of genetic engineering and clinical medicine, and relates to plasma microRNA (miRNA) markers and applications thereof related to the occurrence of common congenital intestinal aganglionosis. Background technique [0002] Common congenital aganglionosis of the bowel (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 mainly characterized by the absence of distal ganglion cells in the digestive tract, which is mainly manifested as enteric nerve migration disorder or abnormal development after migration during the development of the enteric nervous system, resulting in the absence of distal intestinal ganglion cells and impaired distal intestinal function. Finally, the main clinical symptoms are difficulty in defecation, abdominal distension and intestinal obstruction. According to ...

Claims

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Application Information

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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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