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Application of reagent for detecting EAPP genetic expression in Alzheimer disease diagnosis and treatment

A kind of Alzheimer's disease and genetic technology, applied in gene therapy, biological testing, material inspection products, etc., can solve the problems of irreversible disease, unclear etiology and pathogenesis

Active Publication Date: 2015-10-28
QINGDAO MEDINTELL BIOMEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Since the etiology and pathogenesis of AD are still unclear, there is currently no specific method to reverse and prevent the progression of the disease. Therefore, at present, symptomatic treatment is mainly carried out at an early stage, and currently clinically diagnosed AD patients are basically in the middle and late stages, and existing treatments can only improve Symptoms, do not prevent or reverse disease progression

Method used

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  • Application of reagent for detecting EAPP genetic expression in Alzheimer disease diagnosis and treatment
  • Application of reagent for detecting EAPP genetic expression in Alzheimer disease diagnosis and treatment
  • Application of reagent for detecting EAPP genetic expression in Alzheimer disease diagnosis and treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Screening for Gene Markers Related to Alzheimer's Disease

[0055] 1. Collection of peripheral blood samples

[0056] AD patients came from Beijing 301 Hospital, a total of 60 cases, aged 53-84 years old, all cases were diagnosed as AD, and its diagnostic criteria refer to the third revised edition of the American Diagnostic and Statistical Manual of Mental Disorders. A total of 50 cases were selected as the control group, selected from the routine physical examination population of Beijing Hospital. All the subjects were excluded from blood lipid metabolism and other diseases, aged 60-82 years. All subjects signed the informed consent for the testing project and provided peripheral blood for genetic testing.

[0057] 2. Total RNA extraction from blood

[0058] Extraction of total RNA from blood using Biotech Blood RNA Extraction Kit

[0059] (1) Take 250 μl (or 0.25 g) of whole blood into an RNase-Free filter column, centrifuge at 13,000 rpm for 2 minutes,...

Embodiment 2

[0089] Example 2 Construction of EAPP gene expression plasmid

[0090] 1. The human nerve cell line SH-SY5Y was incubated at 37° C. in 5% CO with DMEM (high glucose) medium containing 10% calf serum. 2 , Cultivated in an incubator with a relative humidity of 90%. The medium was changed once every 2-3 days, and 0.25% trypsin was used for routine digestion and passage.

[0091] 2. Overexpression of EAPP gene

[0092] 2.1 Construction of EAPP gene expression vector

[0093] Amplification primers were designed according to the coding sequence of the EAPP gene (as shown in SEQ ID NO.1), and the primer sequences were as follows: the forward primer was 5'-ATGAACCGGCTTCCGGAT-3' (SEQ ID NO.7), and the reverse primer was 5' -TTAGGAATGGCTTGCTAAAAC-3' (SEQ ID NO. 8). The coding sequence of the full-length EAPP gene was amplified from the cDNA library of adult fetal brain (clontech company, product number: 638831), and the above cDNA sequence was double-digested with restriction endonu...

Embodiment 3

[0102] Example 3 Antagonistic effect of EAPP gene expression on neuronal cell death induced by Aβ

[0103] 1. Cell transfection: The neural cell line R2L1 was transfected with pcDNA3.1-EAPP and pcDNA3.1 according to the method in Example 2.

[0104] 2. After 24 hours of transfection, the neural cell line R2L1 was cultured in a 96-well plate with a cell density of 0.5×10 4 cells / well. Divide the cells into the following groups:

[0105] Uninduced group: transfected with pcDNA3.1, without adding 20 μM Aβ42;

[0106] Negative control group (pcDNA3.1+Aβ42): pcDNA3.1 was transfected, and 20 μM Aβ42 was added to the medium at the same time;

[0107] EAPP gene overexpression group (pcDNA3.1-EAPP+Aβ42): transfect pcDNA3.1-EAPP, and add 20 μM Aβ42 to the medium at the same time;

[0108] Three replicate holes were set up for each group. After incubation at 37° C. for 20 h, MTT was added and incubated for 4 h. After adding the dissolving solution, incubate at 37°C for another 12h,...

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Abstract

The invention discloses a molecular marker-EAPP gene for early diagnosis of the Alzheimer disease. An experimental result shows that the mRNA level of the EAPP gene in blood of the patient suffering from the Alzheimer disease is remarkably lower than that of normal people, so that whether a tested person has the risk of the Alzheimer disease or not or whether the tested person has suffered from the Alzheimer disease or not is judged by determining the expression level of the EAPP gene in the blood of the tested person. The EAPP gene can be used for preparation and application for the patient suffering from the Alzheimer disease or the crowd with the high Alzheimer disease risk so as to be used for treating the Alzheimer disease or preventing the Alzheimer disease. The new diagnosis method is provided for diagnosing the Alzheimer disease clinically and the new candidate medicine is provided for treating the Alzheimer disease.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the use of human EAPP gene in the diagnosis and treatment of Alzheimer's disease. Background technique [0002] Alzheimer's disease (AD), also known as senile dementia, is a serious neurodegenerative disease of human health. Clinically, it is characterized by progressive memory loss, cognitive dysfunction and behavioral abnormalities, and its pathological features are brain atrophy, senile plaques and neurofibrillary tangles in the brain, and neuron loss. [0003] Currently known, there are at least 32 types of dementia, among which AD accounts for as much as 50-60% of its incidence. According to the epidemiological survey, there are currently more than 25 million AD patients in the world. In 2008, the number of AD patients in the United States was estimated to reach 5.2 million, and 1 out of 8 people aged 65 and over (13%) was an AD patient, and 1 person was infected with AD every...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C07K16/18C12Q1/68G01N33/53C40B40/08C40B40/10A61K48/00A61P25/28
Inventor 杨承刚李曙光
Owner QINGDAO MEDINTELL BIOMEDICAL CO LTD
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