In vitro method for early detection of severity of aneurysmal subarachnoid haemorrhage and prognosis

A technology for subarachnoid space and cerebral aneurysm, which is applied in the field of medical detection and can solve the problems of inaccurate prediction and inability to predict in advance.

Active Publication Date: 2019-06-11
THE FIRST AFFILIATED HOSPITAL OF WANNAN MEDICAL COLLEGE YIJISHAN HOSPITAL OF WANNAN MEDICAL COLLEGE
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0005] The purpose of the embodiments of the present invention is to provide an in vitro method for early detection of the severity and prognosis of cerebral aneurysmal subarachnoid hemorrhage, so as to solve the problem that the prior art can only judge the patient's consciousness status and head CT bleeding volume. To subjectively judge the condition and lead to inaccurate predictions, defects that cannot be predicted in advance

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  • In vitro method for early detection of severity of aneurysmal subarachnoid haemorrhage and prognosis
  • In vitro method for early detection of severity of aneurysmal subarachnoid haemorrhage and prognosis
  • In vitro method for early detection of severity of aneurysmal subarachnoid haemorrhage and prognosis

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Experimental program
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Embodiment 1

[0030] Embodiment 1 Experimental Design and Specimen Collection

[0031] 1. Experimental design

[0032] All the subjects signed the informed consent form by themselves or their family members, and all the samples used in the research process were approved by the ethics committee. The embodiment of the present invention is divided into three stages: the discovery stage, the coaching group, and the verification stage. Next-generation sequencing was performed on plasma exosomes from brain aneurysmal subarachnoid hemorrhage (aSAH) and the control group, and differential microRNANAs were found. Then, through 10 small samples of plasma, the coaching group's research was carried out to determine the candidate microRNANAs that entered the verification stage. A large sample of candidate microRNANAs was subsequently validated. Neurological prognosis was evaluated by modified Rankin score. Among them, 0-2 is divided into good prognosis, 3-6 is divided into poor prognosis.

[0033] 2...

Embodiment 2

[0035]Example 2 Extraction, electron microscope observation and WB verification of exosomes

[0036] 1. Exosome isolation

[0037] Exosomes were isolated by exoEasy Maxi Kit (from plasma; Qiagen, Valencia, CA). First filter the specimen through a 0.8 μm filter tube. The sample and buffer XBP were mixed in equal volumes 1:1 and incubated at room temperature for 5 minutes. Transfer to a centrifuge tube with a filter membrane, and centrifuge at 500g for 1 minute to remove the centrifugate. The isolated exosomes should be washed with Buffer XWP and centrifuged at 5000g for 5 minutes to remove impurities. Finally, the collected exosomes were lysed with buffer XE. The amount of concentrated exosomes was measured using the BCA method. For transmission electron microscopy, exosomes were absorbed by carbon-coated nickel grids for 1 hour, washed three times with PBS for 5 minutes each time, and fixed with 2% formaldehyde for 10 minutes. Use uranyl acetate and lead citrate for nega...

Embodiment 3

[0040] Example 3 NGS sequencing, data collection and differential microRNAs analysis

[0041] 1. NGS sequencing

[0042] Small RNA library construction: Trizol was used to extract total RNA. Use Bioanalyzer 2100 to quantify and test the purity of total RNA, and the purity is qualified if the RIN is greater than 7.0. About 1ug of total RNA can be used to build a library, and IlluminaHiseq2500 is used for sequencing.

[0043] 2. Data collection

[0044] Raw data were collected by ACGT101-microRNA, and dimers, rRNA, tRNA, snRNA, snoRNA and repetitive sequences were removed. The unique sequences of 18-26 nucleotides were then searched by microRNABase 21.0 to identify known microRNANAs and novel 3p- or 5p-microRNANAs.

[0045] 3. Differential microRNANAs analysis

[0046] Applying Fisher's exact test, X 2 2*2 inspection, X 2 The n*n test, t test or ANOVA based on the experimental design were used to test the difference of the sequencing results. The significance thresholds...

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Abstract

The embodiment of the invention discloses an in vitro method for early detection of the severity of aneurysmal subarachnoid haemorrhage and prognosis, and the method is carried out by measuring the expression level of microRNAs of plasma exosomes. The embodiment of the invention also provides a product for early detection of the severity of aneurysmal subarachnoid haemorrhage and prognosis, and the product determines the severity and prognosis of the aneurysmal subarachnoid haemorrhage by measuring the expression level of the microRNAs of the plasma exosomes. The embodiment of the invention utilizes exosome microRNANA sequencing and real-time PCR method and technology for the first time, screens out potential biomarkers closely related to the occurrence and development of aSAH from blood exosomes, determines that the increased expression of microRNA-193b-3p and microRNA-486-3p is related to poor prognosis and the decreased expression of microRNA-369-3p and microRNA-410-3p is related topoor prognosis, provides a new method for monitoring aSAH and evaluating prognosis, and provides theoretical basis and experimental basis for improving the diagnosis and treatment effect of the disease.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to an in vitro method for early detection of the severity of cerebral aneurysmal subarachnoid hemorrhage, an in vitro method and a product for prognosis. Background technique [0002] Aneurysmal subarachnoid haemorrhage (aSAH) is intracranial subarachnoid hemorrhage caused by cerebral aneurysm bleeding. The clinical mortality and disability rate is as high as 45%, accounting for 5% of stroke. Most of the reasons that affect the prognosis are mainly early brain injury and vasospasm, and the resulting delayed cerebral ischemia. Recently, many researchers have found that early brain injury is the decisive factor leading to the poor prognosis of aSAH, so the research on the early pathophysiological changes of aSAH is particularly important. Early brain damage may be associated with inadequate microcirculation, disruption of ion homeostasis, inflammation, and microvascular co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883
Inventor 赖年升李真保方兴根姚阳赵心同袁金龙刘佳强吴德刚
Owner THE FIRST AFFILIATED HOSPITAL OF WANNAN MEDICAL COLLEGE YIJISHAN HOSPITAL OF WANNAN MEDICAL COLLEGE
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