MYOZ1 gene and application of expression product of MYOZ1 gene in diagnosing and treating parkinsonism

A technology for Parkinson's disease and expression products, which can be used in gene therapy, disease diagnosis, microbial measurement/testing, etc., and can solve problems such as difficulty in popularization and high inspection costs

Active Publication Date: 2015-10-07
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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  • MYOZ1 gene and application of expression product of MYOZ1 gene in diagnosing and treating parkinsonism
  • MYOZ1 gene and application of expression product of MYOZ1 gene in diagnosing and treating parkinsonism
  • MYOZ1 gene and application of expression product of MYOZ1 gene in diagnosing and treating parkinsonism

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 Blood RNA extraction kit from U-gene. Specific steps are as follows:

[0058] 1) Add 5 times the volume of 1×XR-I buffer per volume of fresh blood (maximum 1ml), for example: add 5ml of XR-I buffer per 1ml of blood, and vortex to mix;

[0059] 2) Ice-bathed for 15 minutes, quickly mixed twice on a vortex shaker, the solution became clear, indicating that the red blood cells had been lysed. If the hematocrit or ECR of individual samples increases, extend the ice bath time to 20 minutes;

[0060] 3) Centrifuge at 450g for 10min at 4°C to pr...

Embodiment 2

[0088] Example 2 QPCR sequencing to verify the differential expression of the MYOZ1 gene

[0089] 1. According to the detection results of high-throughput sequencing, the MYOZ1 gene was selected for large-sample QPCR verification. According to the sample collection method in Example 1, 90 cases of blood from Parkinson's patients and 90 cases of normal blood 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. Immediately after 5 minutes in 70°C water bath, ice bath for 2-3 minutes.

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

[0094] (3) After ...

Embodiment 3

[0114] Example 3 Inhibition of MYOZ1 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 1:3-1:4. After 24 hours, the cells enter the logarithmic growth phase and replace the culture medium. Experiments call for different interventions.

[0116] 2. siRNA design

[0117] siRNA sequences against MYOZ1:

[0118] siRNA1-MYOZ1:

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

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Abstract

The invention discloses application of MYOZ1 in diagnosing and treating parkinsonism, belonging to the technical field of new application of genes. The experiment proves that the MYOZ1 gene expression in the blood of a parkinsonism patient is enhanced as compared with a normal person, which indicates that the MYOZ1 expression level in the blood can be detected to diagnose whether a subject suffers from parkinsonism. The in-vitro MTT experiment detect that the MYOZ1 gene expression is not beneficial to growth of human neuroblastoma cells. Therefore, the MYOZ1 has favorable development and application prospects in preparing products for diagnosing and treating parkinsonism.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the use of human MYOZ1 gene in the diagnosis and treatment of Parkinson's disease. Background technique [0002] Parkinson's disease is a common neurodegenerative disease, more common in the elderly, the average age of onset is about 60 years old. The prevalence of Parkinson's in people over 65 years old in my country is about 1.7%. Most Parkinson's patients are sporadic cases, slightly more male than female. The main pathological change of the disease is the degeneration and death of dopaminergic neurons in the substantia nigra of the midbrain, which leads to a significant decrease in the content of dopamine in the striatum and causes the disease. Parkinson's disease is a chronic progressive disease with high heterogeneity and currently there is no cure. In severe cases, the whole body may be stiff, unable to take care of themselves, and even lie in bed for a long time, and event...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/68A61K48/00A61K31/713A61K39/00A61K45/00A61P35/00
CPCA61K31/713A61K39/0005A61K45/00C12Q1/6883C12Q2600/158G01N33/6893G01N2800/2835
Inventor 杨承刚高倩倩
Owner QINGDAO MEDINTELL BIOMEDICAL CO LTD
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