Use of microRNA in exosomes for diagnosis of cerebral arterial thrombosis

A technology of ischemic stroke and exosomes, which is applied in the field of medical diagnosis, can solve the problems of insufficient patients, early and reliable assessment of the cause of stroke, and accurate measurement of personalized treatment plans, etc.

Active Publication Date: 2018-05-25
CHI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] Thus, existing diagnostic techniques cannot provide early and reliable assessment of the cause of stroke, individualized treatment ...

Method used

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  • Use of microRNA in exosomes for diagnosis of cerebral arterial thrombosis
  • Use of microRNA in exosomes for diagnosis of cerebral arterial thrombosis
  • Use of microRNA in exosomes for diagnosis of cerebral arterial thrombosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Research Objects and Serum Collection

[0071] From January 2017 to May 2017, 40 patients with acute stroke in the Department of Neurology, Shenzhen Second People's Hospital were selected as the research group (20 males and 20 males). Among them, there were 20 cases of cardiogenic stroke and 20 cases of non-cardiogenic stroke. Aged 39 to 73 years old, with an average of (56.4±13.3) years old. All of them were confirmed by cranial CT or MRI examination and met the diagnostic criteria of stroke revised by the 4th National Cerebrovascular Disease Academic Conference. The onset time was 48-72 hours, and the median (interquartile range) of NIHSS score was 8 (8-12). In addition to malignant tumors, heart failure, blood, endocrine, metabolic or digestive system diseases, malnutrition, severe lung infection, liver and kidney insufficiency, and other diseases of the nervous system. In the same period, 30 healthy volunteers diagnosed by the physical examination cente...

Embodiment 2

[0074] Example 2 Isolation of Serum Exosomes

[0075] (1) Exosomes in serum were isolated using the Exoquick kit from SBI (System Biosciences Inc., Mountain View, CA, USA). The serum stored at -80°C was thawed on ice and centrifuged at 3000g for 15min. Transfer the supernatant to a new EP tube, add an appropriate amount of ExoQuick solution and mix gently by inversion, react at 4°C for 30 minutes (preferably add 63 μL exoquick reagent to 250 μL serum), and centrifuge at 1500 g for 30 minutes (the exosomes sink under the tube); Aspirate the supernatant, and centrifuge at 1500g for 5 minutes to aspirate all the supernatant (do not shake the centrifuge tube); dissolve all the precipitates with 250 μL PBS, and store at -80°C for later use.

[0076] (2) Analysis of particle size and concentration of exosomes: Nanoparticle-tracking analysis (NTA) was used to analyze the number and size distribution of particles. The above pellet was resuspended with 30 μL particle-free PBS, and th...

Embodiment 3

[0080] Example 3 Extraction of exosome RNA and determination of microRNA by real-time quantitative PCR

[0081] The total RNA of serum exosomes was extracted using mirVana (Ambion, TX, USA) kit. Bioanalyzer2100 (Agilent, CA) detected the quality and concentration of the extracted RNA. The exosomal miRNA in each group was detected by CFX96 PCR instrument (Biorad), and the detection was repeated 3 times for each sample.

[0082] (1) Obtain cDNA by reverse transcription:

[0083] 1) Extract the RNA in the exosomes of the above samples as a template, add template (50-150ng), RT primer (15-20pmol) and RNA free H to the RNase-free PCR tube 2 O to a total volume of 12 μl.

[0084] 2) Mix the above solution and incubate at 65°C for 5 minutes to open the RNA secondary structure, and then place it on ice immediately to prevent RNA renaturation from restoring the secondary structure.

[0085] 3) reverse transcription:

[0086] During the reverse transcription process, the specific r...

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Abstract

The invention discloses the use of an agent for detecting microRNA in exosomes in the preparation of a product for judging the presence and/or degree of cerebral ischemic injury, and relates to a method for early discrimination of a subject suffering from cardiogenic ischemic stroke and non-cardiogenic ischemic stroke. The method uses the exosomes in high-molecular polymer precipitate serum and areal-time fluorescence quantitative PCR technology, and experimental results show that the specificity and sensitivity of miR-155-5p and miR-93-5p are significantly higher than existing protein markers. Meanwhile, miR-133a-3p and miR-208a-3p expression of the exosomes of patients with cardiogenic ischemic stroke is significantly higher compared with patients with non-cardiogenic ischemic stroke under the condition of no significant changes in the serum miR-133a-3p and miR-208a-3p at the early stage so that the agent can be used as a marker to distinguish the cardiogenic ischemic stroke and thenon-cardiogenic ischemic stroke.

Description

technical field [0001] The present invention relates to the field of medical diagnosis, in particular to the use of microRNA in exosomes in diagnosing ischemic stroke, and in particular to the application of microRNA in distinguishing cardiogenic ischemic stroke from non-cardiogenic ischemic stroke . Background technique [0002] After ischemic heart disease, stroke, as a disease with a very high incidence rate in high-income countries, ranks second among the causes of human death announced by the World Health Organization. In terms of disability-adjusted life years, stroke is twice that of coronary heart disease. In addition, the long-term mortality risk of post-stroke patients includes cardiovascular events in addition to stroke recurrence, and the occurrence of long-term cardiovascular events in post-stroke patients increases significantly with the increase in duration. Stroke is a serious public health problem worldwide, and ischemic stroke accounts for 60%-80% of it. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883
CPCC12Q1/6883C12Q2600/112C12Q2600/158C12Q2600/178
Inventor 董鸣
Owner CHI BIOTECH CO LTD
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