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 trans

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

Example Embodiment

[0052] Example 1 Screening of gene markers related to Parkinson's disease

[0053] 1.1 Sample collection

[0054] Collect blood samples of 10 normal people and Parkinson's disease patients. All the above samples were obtained with the approval of the ethics committee.

[0055] 1.2 Preparation and quality analysis of RNA samples

[0056] 1.2.1 Preparation of RNA samples

[0057] Use Promega's RNA extraction kit to extract total RNA. 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 3000 rpm (400 g) for 5 minutes, carefully aspirate the supernatant from the top of the sample;

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

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

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

[0063] 6) Avoid the cell pellets, carefully aspirate alm...

Example Embodiment

[0088] Example 2 QPCR sequencing to verify the differential expression of EPB41L4B gene

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

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

[0091] 3. Reverse transcription: use TAKARA's reverse transcription kit for operation. Specific steps are as follows:

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

[0093] (2) Construct a 25μl reaction system, which includes 5μl of 5× reverse transcription buffer, 5μl of dNTP (2.5mM), RNasin40U / μl, M-MLV200U / μl, and supplement with ribozyme-free water to 25μl;

[0094] (3) After 42℃ water bath for 60 minutes, 95℃ water bath for 5 minutes to inactivate M-MLV;

[0095] (4...

Example Embodiment

[0114] Example 3 Inhibition of EPB41L4B gene expression

[0115] 1. Cell culture: dopamine neuron cells SH-SY5Y, in DMEM / F12 medium (pH7.2~7.4) containing 10% fetal bovine serum, 1% penicillin / streptomycin, at 37℃, 5% CO 2 , Cultivation in an incubator with a relative humidity of 90%. Change the medium every 2 days. Passage when the cells grow to 90% contact, wash with PBS and add 0.25%-EDTA trypsin digestion to separate the cells from the bottle wall, and culture them with DMEM / F12 containing fetal bovine serum The trypsin digestion reaction was terminated by centrifugation at 1000g for 2min, the supernatant was discarded, and the supernatant was resuspended in the newly configured culture fluid, and passaged at a ratio of 1:3~1:4. After 24h, the cells entered the logarithmic growth phase and replaced the culture fluid. Different interventions are required.

[0116] 2. siRNA design

[0117] SiRNA sequence for EPB41L4B:

[0118] siRNA1-EPB41L4B:

[0119] The sense strand is 5’-AAUCUG...

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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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