Gene marker for diagnosis and treatment of Alzheimer's disease
A gene expression and drug technology, used in disease diagnosis, biological testing, drug combination, etc.
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Embodiment 1
[0049] Example 1 QPCR sequencing to verify the expression of PRRT3 gene
[0050] 1. Sample collection
[0051] Blood samples were collected from 49 Alzheimer's patients and 35 healthy controls of the same age. The included Alzheimer's patients met the criteria established by the National Institute of Neurology and the Alzheimer's Disease-Related Diseases Association (NINCDS-ADRDA), and other neurological disorders were excluded through medical history investigation, clinical examination, imaging examination, and routine laboratory examination. Systemic diseases and common geriatric diseases.
[0052] 2. Trizol method to extract RNA from samples
[0053] Mix equal volumes of blood and Trizol evenly, let stand at room temperature for 5 minutes, then add 1 / 5 volume of Trizol reagent in chloroform, mix well, then stand at room temperature for 5 minutes, centrifuge at 12000g at 4°C for 15 minutes, Take the supernatant into a new 1.5mL centrifuge tube, add 0.5-1.0mL / tube of pre-c...
Embodiment 2
[0069] Example 2 Overexpression of PRRT3 gene
[0070] 1. Cell culture
[0071] The SH-SY5Y cell line was cultured in DMEM high glucose complete medium at 37°C, 5% CO 2 , Incubated in an incubator with a relative humidity of 90%.
[0072] 2. Transfection
[0073] 1) Construction of gene overexpression vector
[0074] Specific PCR amplification primers were synthesized according to the sequence of PRRT3 in GeneBank, and two restriction enzyme sites, KpnI and XhoI, were added to the 5' end primer and the 3' end primer, respectively. The cDNA extracted and reverse-transcribed from Alzheimer's patients was used as the amplification template, and the above cDNA sequence was double-digested with restriction endonucleases KpnI and XhoI and then inserted into the eukaryotic cell expression vector cut with KpnI and XhoI In pcDNA3.1(+), the obtained recombinant vector pcDNA3.1(+)-1 was ligated for subsequent experiments.
[0075] 2) Treatment of cells before transfection
[0076] ...
Embodiment 3
[0088] Example 3 Effect of PRRT3 gene on nerve cells
[0089] MTT and apoptosis assays were used to detect the effect of PRRT3 gene on the survival rate of SH-SY5Y Alzheimer's disease cell model cells.
[0090] 1. Cell grouping:
[0091] Blank control group, Aβ 1-40 (16μM Aβ 1-40 Treatment 48h) treatment group, using Aβ 1-40 Treated NC control group and experimental group (pcDNA3.1(+)-1)
[0092] 2. The cell culture and transfection steps are the same as in Example 2.
[0093] 3. MTT detection of cell viability
[0094] Cells were seeded in 96-well plates and cultured overnight. After plating for 1 day, add 15 μL MTT (0.5 kg / mL PBS) to each well and continue to incubate for 4 hours, then add 200 μL DMSO to each well, shake for 10 minutes, and place at room temperature for 10 minutes to make blue-purple crystals substance fully dissolved. Calibrate to zero with a blank well, and use a microplate reader to measure the absorbance of each well at a wavelength of 570 nm. Th...
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