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Application of EPB41L4B gene in diagnosis and treatment of Parkinson disease

A Parkinson's disease, gene technology, applied in the treatment, the human EPB41L4B gene in the field of Parkinson's disease diagnosis, can solve the problems of difficult promotion, expensive examination and other problems

Active Publication Date: 2016-02-17
QINGDAO MEDINTELL BIOMEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Auxiliary detection methods for Parkinson's disease, such as 99mTc-TRODAT-1, can be used as a tracer to perform functional imaging of dopamine transporters to support the diagnosis, but the inspection costs are relatively expensive, and it is difficult to promote large-scale clinical application

Method used

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  • Application of EPB41L4B gene in diagnosis and treatment of Parkinson disease
  • Application of EPB41L4B gene in diagnosis and treatment of Parkinson disease
  • Application of EPB41L4B gene in diagnosis and treatment of Parkinson disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Screening for gene markers associated with Parkinson's disease

[0053] 1.1 Sample collection

[0054] 10 normal blood samples and blood samples from patients with Parkinson's disease were collected, and all the above samples were obtained with the consent of the ethics committee.

[0055] 1.2 RNA sample preparation and quality analysis

[0056] 1.2.1 Preparation of RNA samples

[0057] Total RNA was extracted using the RNA extraction kit from Promega. Specific steps are as follows:

[0058] 1) Take 1ml of whole blood collected in a test tube treated with heparin or EDTA, and put it into a sterile centrifuge tube;

[0059] 2) Collect blood cells: centrifuge at 3000rpm (400g) for 5min, carefully suck off the supernatant from the top of the sample;

[0060] 3) Add 1ml of blood cell lysate, pipette carefully 4-5 times, and resuspend the sediment;

[0061] 4) Centrifuge at 3000rpm for 5min;

[0062] 5) Repeat steps 3), 4) twice (three times in total);

[0...

Embodiment 2

[0088] Example 2 QPCR sequencing verification of differential expression of EPB41L4B gene

[0089] 1. According to the detection results of high-throughput sequencing, the EPB41L4B gene was selected for large-sample QPCR verification. According to the sample collection method in Example 1, 80 cases of blood from Parkinson's patients and blood from normal people were selected.

[0090] 2. The RNA extraction steps are the same as in Example 1.

[0091] 3. Reverse transcription: use the reverse transcription kit of TAKARA company to operate. Specific steps are as follows:

[0092] (1) Take 2 μg of total RNA for reverse transcription, add 2 μl of Oligo(dT) and mix well; 70°C water bath; 5 min and then immediately ice bath for 2-3 min;

[0093] (2) Construct a 25 μl reaction system, including 5 μl of 5× reverse transcription buffer, 5 μl of dNTP (2.5 mM), RNasin 40 U / μl, M-MLV 200 U / μl, and supplement nuclease-free water to 25 μl;

[0094] (3) After 42°C water bath for 60 minut...

Embodiment 3

[0114] Embodiment 3 suppresses EPB41L4B gene expression

[0115] 1. Cell culture: dopamine neuron cells SH-SY5Y, in DMEM / F12 culture medium containing 10% fetal bovine serum and 1% penicillin / streptomycin (pH7.2~7.4), at 37°C, 5% CO 2 , Cultivated in an incubator with a relative humidity of 90%. Change the medium once every 2 days, and subculture when the cells grow to 90% contact, wash with PBS, add 0.25%-EDTA trypsin to digest the cells from the bottle wall, and culture with DMEM / F12 containing fetal bovine serum solution to stop the trypsin digestion reaction, centrifuge at 1000g for 2min, discard the supernatant, resuspend with the newly prepared culture medium, and passage at a ratio of 1:3 to 1:4. After 24 hours, the cells enter the logarithmic growth phase and replace the culture medium. Different interventions are required.

[0116] 2. siRNA design

[0117] siRNA sequence against EPB41L4B:

[0118] siRNA1-EPB41L4B:

[0119] The sense strand is 5'-AAUCUGAUCAAAACAAA...

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Abstract

The invention discloses application of an EPB41L4B gene in diagnosis and treatment of a Parkinson disease. It is proved by experiments that compared with a normal person, expression of the EPB41L4B gene in the blood of a Parkinson disease patient is up-regulated, and it is shown that whether a subject suffers from the Parkinson disease or not can be diagnosed according to the expression level of EPB41L4B in the blood. According to the research results of the application of the EPB41L4B gene in the diagnosis and treatment of the Parkinson disease, the EPB41L4B can further be used for preparing a drug for treating the Parkinson disease. According to the application of the EPB41L4B gene in the diagnosis and treatment of the Parkinson disease, the sensibility and the specificity of Parkinson disease diagnosis are greatly improved, and meanwhile a new target is provided for gene treatment of the Parkinson disease.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of human EPB41L4B gene in the diagnosis and treatment of Parkinson's disease. Background technique [0002] Parkinson's disease (PD) is a common progressive neurodegenerative disease in middle-aged and elderly people, clinically characterized by tremor, bradykinesia, muscle rigidity, and abnormal posture and gait. Pathological features include not only degeneration of the nigrostriatal dopaminergic system causing core dyskinesia symptoms, but also multitarget invasion of the central, peripheral, and autonomic nervous systems with extensive Lewy bodies and Lewy axons form. The cause of neuronal cell death in the substantia nigra in Parkinson's disease is not completely clear, and it is generally believed that it is related to many mechanisms, such as environmental toxins, gene mutations, genetic factors, oxidative stress, abnormal immune system, iron ion accumulation,...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/68A61K45/00A61K48/00A61K31/7088A61P25/16
Inventor 董琳杨承刚边洋
Owner QINGDAO MEDINTELL BIOMEDICAL CO LTD
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