Hypoxic-ischemic brain damage diagnosis target and application

A technology for hypoxia-ischemia and brain injury, which is applied in the field of disease detection, can solve the problems of timely and effective diagnosis during pregnancy and other problems, and achieve the effect of accurate detection method and high detection accuracy

Active Publication Date: 2019-06-21
BEIJING FRIENDSHIP HOSPITAL CAPITAL MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above diagnosis cannot be ti

Method used

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  • Hypoxic-ischemic brain damage diagnosis target and application
  • Hypoxic-ischemic brain damage diagnosis target and application
  • Hypoxic-ischemic brain damage diagnosis target and application

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0035] The establishment and sampling of the hypoxia-ischemia rat model of embodiment 1

[0036] 7-day-old neonatal SD rats with a body weight of 10-14 g. They were randomly divided into two groups, a sham operation group (normal control group) and a hypoxic-ischemic group, with 3 rats in each group. Under ether anesthesia, a median neck incision was made, and the left common carotid artery was separated. The sham operation group did not receive any treatment; the operation group ligated and cut off the left common carotid artery, rested for 1 hour, and placed in 8% oxygen and nitrogen mixed gas for 2 hours. One day after the experiment, the rats in each group and at each time point were decapitated under ether anesthesia, and the brain tissues were taken out and immediately put into liquid nitrogen for cryopreservation.

Embodiment 2

[0037] Example 2 High-throughput sequencing and data analysis

[0038] 2.1 Extraction and quality inspection of tissue total RNA

[0039] (1) Cut the tissues of each group into pieces on ice, take an appropriate amount (50-100 mg) of the tissues, add 1 ml of pre-cooled Trizol to the EP tube without RNase, pipette evenly, and let stand on ice for 10 minutes;

[0040] (2) Add 200 μl of pre-cooled chloroform, shake vigorously for 30 sec, then let stand on ice for 15 min, and centrifuge at 14000 rpm / min at 4°C for 20 min;

[0041] (3) Take out the supernatant and put it in a new RNase-free EP tube (be careful not to touch the middle layer), add an equal volume of pre-cooled isopropanol (about 500 μl), mix well and let stand on ice for 0.5-1h, 4 Centrifuge at 14000rpm / min for 20min;

[0042] (4) Discard the supernatant, add 1 ml of pre-cooled 75% ethanol, mix upside down, and centrifuge at 14000 rpm / min at 4°C for 20 min;

[0043] (5) Discard the supernatant, add 1 ml of pre-coo...

Embodiment 3

[0063] Example 3 RT-PCR verification of the relative expression of circRNA in the control group and the model group

[0064] 1. Model building

[0065] Concrete method is with embodiment 2. The sham operation group (normal control group) and the hypoxia-ischemia group had 20 rats in each group.

[0066] 2. Experimental method

[0067] 2.1 Extraction of total tissue RNA

[0068] Reference Example 2

[0069] 2.2 Primer design

[0070] For the circular closed structure of circRNA, design back-to-back primers (divergent primer), GAPDH is used as an internal reference, and sent to a primer synthesis company for synthesis.

[0071] circRNA primers:

[0072] Upstream primer: 5'-AAGCCAGGGGAACACTAGTC-3' (SEQ ID NO.2)

[0073] Downstream primer: 5'-GTTAAGTTCCAATGCCGGCC-3' (SEQ ID NO.3)

[0074] Target gene amplification length: 96bp.

[0075] 2.3 Reverse transcription

[0076] According to PimeScript TM RT reagent Kit (Perfect Real Time) RR037A (Takara) manual configures the ...

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Abstract

The invention relates to a hypoxic-ischemic brain damage diagnosis target and application. Hypoxic-ischemic encephalopathy of the newborn is one of the common reasons of child nervous system impairment, and no effective diagnosis methods for the hypoxic-ischemic encephalopathy exist currently. The invention performs circRNA transcriptome sequencing on a hypoxic-ischemic brain damage model, discovers 9 circRNA closely related to the hypoxic-ischemic brain damage through bioinformatics analysis and verifies the 9 circRNA. The invention provides a potential biological marker for the diagnosis ofthe hypoxic-ischemic brain damage and lays a foundation for the clinical gene detection of the hypoxic-ischemic brain damage.

Description

technical field [0001] The invention belongs to the field of disease detection, and in particular relates to a molecular diagnosis target for hypoxic-ischemic brain injury and its application. Background technique [0002] Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the common causes of neurological damage in children. This disease is brain damage caused by perinatal asphyxia and hypoxia. Obstacles that reduce the blood oxygen concentration can cause suffocation, and severe suffocation can cause permanent neurological damage. At present, clinical manifestations are the main basis for diagnosing HIE, and the following four conditions can be confirmed: 1) There is a clear history of abnormal obstetrics that can cause fetal distress, and severe manifestations of fetal distress (fetal heart rate less than 100 beats per hour) min, lasted for more than 5 minutes; and / or amniotic fluid degree III contamination), or obvious asphyxia during delivery; 2) severe asphyxia ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/113
Inventor 杨丽君崔红赵贺华张囡
Owner BEIJING FRIENDSHIP HOSPITAL CAPITAL MEDICAL UNIV
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