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Determination of biological tag-earthworm in-vivo P450 content

The technology of a biomarker and a determination method is applied in the field of the determination of the content of p450 in the body of a biomarker-earthworm, which can solve the problems such as no mature method, and achieve the effects of ensuring effective removal, improving efficiency and reducing cost.

Inactive Publication Date: 2006-03-15
SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are still no reports of mature methods in the world

Method used

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  • Determination of biological tag-earthworm in-vivo P450 content

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020]1) Pretreatment: Wash the living earthworms used in the experiment with distilled water, blot them dry with filter paper, and weigh them; place them in moist filter paper at room temperature for 24 hours to drain the substances in the body before use.

[0021] 2) Preparation of microsomes: Soak live earthworms in 20% glycerin solution at 4°C for 1 hour after the substances in the body have been removed, transfer them into 0.15M KCl solution, divide and break the whole earthworms, and place them in a filter device for use. Wash the blood in the body with 0.15M KCl solution until the cleaning solution is colorless, then transfer it to a 25ml centrifuge tube containing 6ml of homogenization buffer, and homogenize with an internal cutting tissue homogenizer at 8000 rpm for 50 seconds , put the homogenate into a 10ml centrifuge tube, centrifuge at 15,000g for 30min on a low-temperature (4°C) ultracentrifuge, transfer the supernatant to a 10ml centrifuge tube again, and centrif...

Embodiment 2

[0030] The difference from Example 1 is that the earthworm body is placed in filter paper for 24 hours; the homogenization time is 30 seconds in 0.10M KCl solution, and the ultracentrifuge is operated at a low temperature of 3°C, with a centrifugal force of 12000g and a centrifugation time of 30min. The primary centrifugal force is 120000g, and the secondary centrifugal time is 120min; carbon monoxide is introduced for 2min, and left for 2min; the results are shown in Table 1, the sample numbers are, sample 12 and sample 13, and the control group 11, wherein the operating conditions of the control group are the same as the sample group, and the control group The group refers to the earthworms cultivated under the condition of non-polluted soil, and the sample group refers to the earthworms cultivated in the polluted soil.

Embodiment 3

[0032] The difference from Example 1 is that the earthworm body is placed in the filter paper for 36 hours; the homogenization time in 0.12M KCl solution is 40 seconds, and the ultracentrifuge is operated at a low temperature of 3 ° C. The first centrifugal force is 14000g, and the first centrifugal time is 25min. The primary centrifugal force was 140000g, and the secondary centrifugal time was 100min; carbon monoxide was introduced for 2min and left for 3min; the results were shown in Table 1, the sample numbers were, sample 22 and sample 23, and the control group 21, wherein the operating conditions of the control group were the same as those of the sample group, and the control group The group refers to the earthworms cultivated under the condition of non-polluted soil, and the sample group refers to the earthworms cultivated in the polluted soil.

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Abstract

A method for determining P450 content in biological tag ¿C earthworm body includes soaking earthworm living body in glycerin then moving it into salt solution for breaking it to be pieces, moving broken matter into homogenizing solution for homogenizing of 30 ¿C 50 second at 6000 ¿C 8000 rpm, then centrifugalizing homogenized matter for 20 ¿C 30 min at 12000 ¿C 15000 g under 3 ¿C 5 Deg C.; removing off supernatant then centrifugalizing for 90 ¿C 120 min at 120000 ¿C 150000g, discarding supernatant and putting particles in ice water, removing off red matter; dissolving particle in buffer solution and taking out 1 ¿C 2 mI for detecting protein content as the rest for being used to determine P450 content .

Description

technical field [0001] The invention relates to cytochrome P-450, in particular to a biomarker-a method for measuring the content of p450 in earthworms. Background technique [0002] Cytochrome P-450 (CYP) is a kind of metabolic enzyme system with important physiological functions that widely exists in different organisms such as animals, plants and microorganisms, and is widely used in clinical medicine, medicine and other fields. For example, the cytochrome P-450 (CYP) enzyme system can catalyze a large amount of lipophilic foreign substances, and undertake the metabolism and detoxification of a large number of liver swelling drugs (document 2. Shen Jun, Xu Peipei, Jin Xipeng. 1997. The method for the determination of cytochrome P450 in the liver Improvement. Industrial Hygiene and Occupational Diseases, 23(4): 236-238; Literature 3. Leng Xinfu, Qiu Xinghui. 2001. The structure, function and application prospect of cytochrome P450 enzyme system. Science Press. P2; Literatu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/33G01N33/48G01N1/28
Inventor 宋玉芳张薇刘淼
Owner SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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