Inducing and preparation method of rat liver S9

A technology of inducer and heparin, applied in the field of protease, can solve problems such as environmental pollution, and achieve the effect of increasing enzyme content and increasing yield

Inactive Publication Date: 2017-06-13
JIANGYIN CHI SCI
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Problems solved by technology

The traditional rat liver S9 inducer is polychlorinated biphenyl, and its induction effect is better than that of phenobarbital sodium or β-naphthone alone, but it is a carcinogen and a serious environmental pollutant. Prohibition of production

Method used

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  • Inducing and preparation method of rat liver S9
  • Inducing and preparation method of rat liver S9
  • Inducing and preparation method of rat liver S9

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Embodiment Construction

[0020] In order to present the purpose and advantages of the present invention more clearly, specific embodiments will now be further described. The specific embodiments described here are only for explaining the present invention, and are not intended to limit the present invention.

[0021] a. Prepare a basin of -80°C ice before the experiment, place the required liver lavage buffer and storage buffer on the ice 30 minutes in advance, and place several weighed 50mL centrifuge tubes on the ice at the same time.

[0022] b. Select SD rats, male, 230±20g; induction method 1: Rats are intraperitoneally injected with sodium phenobarbital, once a day, the dose is 30 mg / kg on the first day, and 60 mg / kg per day for the remaining 3 days; 3, 4 days intraperitoneal injection of β-naphthoflavone: once a day, the dose is 80mg / kg; Induction method 2: On the 1st day, rats are injected intraperitoneally with phenobarbital sodium at a dose of 30mg / kg and β-naphthol at a dose of 40mg / kg -Na...

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Abstract

The invention provides an inducing and preparation method of rat liver S9. The inducing and preparation method comprises the following steps of (a) selecting a rat, injecting two types of inducing agents into an abdominal cavity by different types and dosages according to the weight of the rat, and determining the optimum inducing method; (b) killing the rat, removing residual blood in the livers under the sterile condition, and fetching out the livers; (c) under the sterile condition, adding a pre-freezing storage buffer liquid according to a certain liver-wet weight ratio, fully shearing, and equalizing pulp in an ice bath; (d) centrifuging a tissue liquid after pulp equalizing at low speed, collecting a supernatant and a precipitate, centrifuging the supernatant liquid at high speed, and collecting the supernatant liquid, so as to obtain the liver S9 for the first time; (e) adding the pre-freezing storage buffer liquid into the collected precipitate according to a certain ratio, fully grinding under the sterile and ice bath conditions, crushing cells by ultrasonic waves under different conditions, centrifuging at high speed, and collecting the supernatant liquid, so as to obtain the liver S9 again; (f) detecting the contents of protein and enzyme in the liver S9 by a BCA (bicinchoninic acid) method and a CO (carbon monoxide) reduction method under the different conditions. The inducing and preparation method of the rat liver S9 has the advantages that the output of liver S9 is increased, and the content of enzyme P450 is obviously increased.

Description

technical field [0001] The invention belongs to the field of protease, in particular to a method for inducing and preparing rat liver S9. Background technique [0002] Rat hepatocyte S9 is the mitochondria-free supernatant of rat liver parenchymal cell homogenate, which contains a large number of drug-metabolizing enzymes such as CYPs. Because of the characteristics of quantitative screening, it is often used as a tool for studying drug metabolism and drug interaction in vitro. At the same time, the biotransformation of many carcinogens depends on the mixed-function oxidase system of intracellular microsomes, so rat liver S9 is the activation system for many in vitro studies on the biotransformation of carcinogens. The traditional rat liver S9 inducer is polychlorinated biphenyl, and its induction effect is better than that of phenobarbital sodium or β-naphthone alone, but it is a carcinogen and a serious environmental pollutant. Prohibition of production. Existing studie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C07K14/47C12N9/02
CPCC07K14/47C12N5/067C12N9/0036C12N9/0042C12N9/0083C12Y106/02004
Inventor 田瑞张亚洲蒋敏齐来俊
Owner JIANGYIN CHI SCI
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