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High-flux evaluation method for cholesterol and aliphatic acid metabolism inhibition medicament

A technology of fatty acid metabolism and evaluation method, which is applied in the field of high-throughput evaluation of cholesterol and fatty acid metabolism-inhibiting drugs, can solve the problems of not having the conditions for high-throughput drug screening, not having the advantage of being widely used, and high requirements for instruments and equipment, Achieve high product extraction efficiency, improve market competitiveness, and facilitate operation.

Active Publication Date: 2019-04-16
辉源生物科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has high requirements for equipment and does not have the advantage of being widely used
However, traditional cholesterol and fatty acid extraction methods are also limited in the amount of materials used, and do not have the conditions for high-throughput drug screening.

Method used

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  • High-flux evaluation method for cholesterol and aliphatic acid metabolism inhibition medicament
  • High-flux evaluation method for cholesterol and aliphatic acid metabolism inhibition medicament

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Culture and labeling of HepG2 cells:

[0035] Cells were seeded in a 96-well cell culture plate with 10,000 cells per well. The medium is a complete medium containing 10% fetal bovine serum and 25 mM glucose. After 48 hours, replace with fresh complete medium. After 18 hours, 200 μl per well was replaced with fresh medium (10% fetal bovine serum, 5.5 mM glucose and no pyruvate). Candidate compounds 1, 2, 3, 4 were added for 1 hour. Add C14-labeled pyruvate (2 μCi) to the corresponding wells, and label for 5 hours at 37° C., 5% CO2.

[0036] Determination of C14-labeled pyruvate incorporation in the cholesterol synthesis pathway:

[0037] Transfer the above cell cultures to new 96-well polypropylene deep-well plates. At room temperature, 200 μl of 0.1N NaOH was added to the cells to lyse the cells, and the cell lysate and culture medium were combined. Add 68 μl of 50% (W / V) KOH to the above mixture, seal with parafilm and cover. Put the 96-well deep-well plate con...

Embodiment 2

[0041] Culture and labeling of A549 cells:

[0042] Cells were seeded in a 96-well cell culture plate with 10,000 cells per well. The medium is a complete medium containing 10% fetal bovine serum and 25 mM glucose. After 48 hours, replace with fresh complete medium. After 18 hours, 200 μl per well was replaced with fresh medium (10% fetal bovine serum, 5.5 mM glucose and no pyruvate). Candidate compounds 5, 6, 7, and 8 were added for 1 hour. Add C14-labeled pyruvate (2 μCi) or acetic acid (1 μCi) to the corresponding wells, and label for 5 hours at 37° C., 5% CO2.

[0043] Determination of the incorporation of C14-labeled pyruvate and acetic acid in the cholesterol synthesis pathway:

[0044] Transfer the above cell cultures to new 96-well polypropylene deep-well plates. At room temperature, 200 μl of 0.1N NaOH was added to the cells to lyse the cells, and the cell lysate and culture medium were combined. Add 68 μl of 50% (W / V) KOH to the above mixture, seal with parafil...

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Abstract

The invention relates to a high-flux evaluation method for a cholesterol and aliphatic acid metabolism inhibition medicament. The method comprises the following steps that (1) cells are cultured and marked; (2) cholesterol is extracted in the alkaline environment via petroleum ether, a Microbeta liquid scintillation counting instrument is used for counting, and an cholesterol metabolism inhibitioneffect of the inhibition medicament to be evaluated is obtained; and (3) aliphatic acid is extracted in the acidic environment via the petroleum ether, the Microbeta liquid scintillation counting instrument is used for counting, and an aliphatic acid metabolism inhibition effect of the inhibition medicament to be evaluated is obtained. Compared with the prior art, a simple, rapid, high-efficiencyand high-flux cholesterol and aliphatic acid extraction technology is established, the cost of instrument is reduced, convenience is provided for operation, the product extraction efficiency is higher, and the method is much more competitive in the market.

Description

technical field [0001] The invention relates to the technical field of tracing, in particular to a high-throughput evaluation method for drugs that inhibit cholesterol and fatty acid metabolism. Background technique [0002] Cholesterol is widely present in animals, especially in brain and nerve tissue, and is also high in kidney, spleen, skin, liver and bile. Cholesterol is an indispensable and important substance in animal tissue cells. It not only participates in the formation of cell membranes, but also is a raw material for the synthesis of bile acids, vitamin D and steroid hormones. Cholesterol is also an important indicator of clinical biochemical examination. Under normal circumstances, the cholesterol synthesized by the body in the liver and taken in from food will be converted into steroid hormones or become components of cell membranes, and keep the concentration of cholesterol in the blood constant. . When severe lesions occur in the liver, the cholesterol conc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50
CPCG01N33/5008
Inventor 朱建梁刘长伟严中华
Owner 辉源生物科技(上海)有限公司
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