Novel serology biomarker GSK3beta detection method for cognitive impairment of diabetic

A technology for diabetes and patients, applied in the field of detection of a new serological marker-GSK3β, which can solve the problems of unsatisfactory specificity and sensitivity, and achieve the effect of simple operation

Inactive Publication Date: 2015-06-17
HUAZHONG UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It has been reported in the literature that there are changes in the blood of AD patients such as apolipoprotein E, platelet APP isoform ratio, homocysteine, miRNA, interleu

Method used

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  • Novel serology biomarker GSK3beta detection method for cognitive impairment of diabetic
  • Novel serology biomarker GSK3beta detection method for cognitive impairment of diabetic
  • Novel serology biomarker GSK3beta detection method for cognitive impairment of diabetic

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Dot-blot method to detect platelet GSK-3β activity in diabetic patients

[0041] 1. Platelet separation and purification.

[0042] 1.1 Human whole blood 5ml, 60g, centrifuged at 4 degrees Celsius for 15 minutes, the upper layer is plasma, the middle layer is white blood cells, and the lower layer is red blood cells. Plasma, 60g, centrifuged at 4°C for 15min, to remove sediment, centrifuged at 1500g for 15min, divided into 2 layers, the upper layer was serum, and the lower layer was platelets.

[0043] 1.2 Platelet samples were washed with modified Tyrode's solution, centrifuged at 1500g for 5min three times, and stored in a -80°C refrigerator.

[0044] 2. Platelet protein extraction and concentration determination.

[0045] 2.1 Add pre-cooled lysate 120μl 2×Buffer to platelets, said 2×Buffer consists of 50mM Tris-Cl pH 8.0, 1% NP-40, 150mM NaCl, 0.1% SDS, 0.02% NaNR3R, 20% glycerol, cocktail, Composed of 1% PMSF, after standing on ice for 20 minutes, the p...

Embodiment 2

[0054] Example 2: Enzyme activity kit detects platelet GSK-3β activity in diabetic patients

[0055] 1. Preparation of samples to be tested

[0056] 1.1 Prepare the purified platelet sample, carefully add 3 ml of GENMED cleaning solution (Reagent A), and mix well. Transfer to a pre-chilled 10ml tube. Centrifuge at 4°C for 5 minutes at 300g. Carefully aspirate the supernatant.

[0057] 1.2 Add 500 microliters of GENMED Lysis Solution (Reagent B) and mix well. Transfer to a pre-chilled 1.5 ml centrifuge tube. Vortex vigorously for 15 seconds. Incubate on ice for 30 minutes. Centrifuge at 4°C for 5 minutes at a speed of 16,000 g.

[0058] 1.3 Carefully pipette 500 µl of the supernatant into a new pre-cooled 1.5 ml centrifuge tube. Pipette 10 μl for protein quantification. Immediately store at -70°C or place in an ice bath to continue subsequent operations.

[0059] 2. Assay Preparation

[0060] 2.1 Place the platelet sample in an ice bath.

[0061] 2.2 Set the conditi...

Embodiment 3

[0079] Experimental study on the role of platelet GSK-3β activity in cognitive impairment in patients with type 2 diabetes mellitus (T2DM)

[0080] Experimental methods: (1) Inclusion criteria: Diabetic patients aged >50 years old; no visual and hearing impairments that significantly affect cognitive tests; no history of severe hypoglycemia, diabetic ketosis coma, hyperosmolar coma; no alcohol dependence and psychoactivity History of substance abuse; no brain trauma, no cardiovascular and cerebrovascular events, including cerebral infarction, cerebral hemorrhage, TIA, myocardial infarction, etc.; no other serious physical diseases that affect cognitive tests, such as epilepsy, hypothyroidism, hypoxemia symptoms, etc.; no history of mental illness; voluntary participation in this study, able to cooperate with the examination, and good compliance.

[0081] (2) Study groups: type 2 diabetes without cognitive impairment T2DM group (T2DM), type 2 diabetes with mild cognitive impair...

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Abstract

The invention provides a novel serology biomarker GSK3beta detection method for cognitive impairment of a diabetic. The method comprises an enzyme activity assay method and a relative quantification dot-blot method. According to the enzyme activity assay method, a GSK-3beta enzyme activity assay kit of GENMED is applied; GSK-3beta phosphorylation is performed under the suppression of GSK-3alpha; along with oxidation reaction of reduced form of nicotinamide-adenine dinucleotid (NADH), a pyruvate kinase and lactic dehydrogenase continuous circulation method reaction system adopts the spectrophotometric method to measure the peak value change after oxidization so as to reflect GSK 3beta activity in a sample. The dot-blot method transfers blood platelet protein to an NC film according to the antigen and antibody fixation reaction principle and then utilizes antibody to perform detection. The novel serology biomarker GSK3beta detection method is used for studying GSK-3beta protein and enzyme activity expression in the diabetic blood and is expected to become a serology biomarker for early diagnosis of Alzheimer's disease (AD).

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a detection method of a new serological marker-GSK3β for cognitive impairment of diabetic patients. Background technique [0002] Type 2 diabetes mellitus (T2DM) and Alzheimer's disease (Alzheimer's disease, AD) are both diseases related to aging. Recent studies have shown that T2DM can significantly increase the risk of AD. Some scholars also call AD For "type 3 diabetes". T2DM is characterized by insulin resistance, insulin secretion disorders and increased glucose production, while abnormal insulin and blood glucose levels in the brain are important causes of AD. The typical pathological features of AD are the formation of neurofibrillary tangles mainly composed of hyperphosphorylated microtubule-associated protein tau in a large number of neurons and the deposition of β-amyloid polypeptide and the formation of a large number of senile plaques outside the neuro...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/6896
Inventor 王建枝徐志鹏
Owner HUAZHONG UNIV OF SCI & TECH
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