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Method for measuring sensitivity and positioning of cholesterol in cells and diagnostic reagent thereof

A measurement method, cholesterol technology, applied in biochemical equipment and methods, disease diagnosis, biological testing, etc., to achieve the effect of important clinical application value

Inactive Publication Date: 2018-03-16
阮雄中
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  • Summary
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  • Description
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AI Technical Summary

Problems solved by technology

[0015] There is a "blind spot" in the traditional method of solely using blood cholesterol as an indicator of cardiovascular and cerebrovascular risk assessment

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0035] Preparation of LDL standards

[0036] Take plasma from healthy volunteers, add antioxidants (100umol / L EDTA, 20umol / L butylated hydroxytoluene), centrifuge below 10000g for 30min to remove chylomicrons, then add sodium bromide to adjust the plasma density to 1.006kg / L, 40000r / Min ultracentrifugation for 18 hours, suck out the upper layer milky white liquid (VLDL) and the second layer light yellow liquid (IDL), collect the lower layer liquid, then add sodium bromide (NaBr) to adjust its final density to 1.063gkg / L, and then ultracentrifuge again at 40000r / min After 24 hours, the upper layer of orange-yellow liquid was sucked out to obtain LDL.

[0037] cell culture method

[0038] Separate PBMCs from healthy people and diseased people, and a small part of the cells are directly used to detect the expression level of the cholesterol sensor SCAP in the PBMCs; most of the cells are planted in 12-well plates with RPMI1640 medium containing 10% fat-free serum, Cultured for...

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Abstract

The invention discloses a method for measuring the sensitivity and positioning of cholesterol in cells and a diagnostic reagent thereof. The method comprises four steps: (1) separating fresh PBMC cells, carrying out smearing and fixing free cholesterol; (2) carrying out specific dual fluorescent probe incubation of endoplasmic reticulum and mitochondria; (3) performing dual probe labeling; and (4)quantifying and correcting fluorescence in cells. The results show that the content of cholesterol in blood is not in a definite positive relationship with the degree of atherosclerosis and the concentration of cholesterol in tissues of aorta and coronary artery. In a human body, there are multiple cholesterol pools; under the action of a certain pathogenic factor, cholesterol is redistributed with respect to organelle; and thus compared with measurement on the concentration of cholesterol in blood, it is more important to detect and position the cholesterol level in cells.

Description

technical field [0001] The invention relates to a method for measuring intracellular cholesterol sensitivity and localization and its diagnostic reagent. Background technique [0002] Cardiovascular and cerebrovascular diseases are the number one killer of human health, and blood lipid metabolism disorders, especially elevated plasma cholesterol levels, are currently recognized as important risk factors for arteriosclerosis and cardiovascular and cerebrovascular diseases. Therefore, detection of plasma cholesterol levels has become a clinical practice. One of the most widely used risk assessment methods in the world. However, we cannot ignore the fact that clinically many patients are not high in blood lipids and are not obese, but they still suffer from arteriosclerosis (myocardial infarction); in addition, people with other diseases, such as chronic kidney disease, diabetes, autoimmune Diseases (systemic lupus erythematosus, etc.), etc. These patients are characterized by...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/92G01N33/50C12Q1/6883
CPCC12Q1/6883C12Q2600/158G01N33/5005G01N33/92G01N2800/32
Inventor 阮雄中
Owner 阮雄中
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