CN102156055A - Method for quickly extracting prelarval fish steroid hormones - Google Patents

Abstract

The invention relates to a method for quickly extracting prelarval fish steroid hormones. The technical scheme of the method comprises the following steps of: firstly cutting weighed prelarval fish; then placing the cut prelarval fish in a mortar containing anhydrous ethyl alcohol and grinding; and pulverizing with ultrasonic waves; centrifuging homogenates to obtain a supernatant; adding dichloromethane of 5 times of the supernatant by volume; uniformly and repeatedly mixing, then standing and dimixing; removing a water phase of an upper layer; placing an organic phase of a lower layer into a water bath of 45 DEG C, and volatizing the organic phase completely; and finally, dissolving with a PBS (Phosphate Buffered Saline) buffer solution containing 1% BSA (Bovine Serum Albumin), and storing at -20 DEG C for later use. The method for quickly extracting the prelarval fish steroid hormones, provided by the invention, can be used for extracting various steroid hormones of prelarval fish and is suitable for control and research of neuroendocrine of various fishes.

Description

A kind of young fast juvenile fish steroid hormone extracting method

Technical field

The present invention relates to the extractive technique of fish steroid hormone.

Background technology

Steroid hormone is the similar lipophilicity micromolecule of chemical constitution, mainly comprises: female hormone, male sex hormone, corticosteroid hormone etc.They are water insoluble relatively, and the plasma membrane that is easy to pass target cell enters in the cell, form hormone-receptor complex with receptors bind in tenuigenin and the nucleus, and regulatory gene is expressed, and influences the growth and the differentiation of particular tissues.In fish,, can also study the influence that external environment is grown to fish self simultaneously by detecting growing and regulatory mechanism of its level of respectively organizing steroid hormone energy researching fish.

At present, for adult fish,, thereby be the main object that steroid hormone is measured because its serum amount that can obtain is more.And serum content is few in individual less fish and the young juvenile fish, is difficult to carry out the research and analysis of steroid hormone.About the mensuration and the domestic report of not seeing as yet of regulatory mechanism research of steroid hormone level in the young juvenile fish of fish, and also only see the report of measuring estradiol content in summer lefteye flounder and the goby juvenile fish by the method for ether extracting abroad.

Summary of the invention

The problem that the present invention need solve has provided a kind of young juvenile fish steroid hormone extracting method of simple and fast.

Technical scheme of the present invention utilizes ethanol and methylene chloride that it is dissolved young juvenile fish historrhexis again, and concrete steps are as follows:

Step 1, get fresh or-20 ℃ of frozen young juvenile fish, shred after the weighing and place mortar;

Step 2, in mortar, adds 50% ethanol, fully after the grinding homogenate is changed in the centrifuge tube according to the ratio of heavy corresponding 1ml 50% ethanol of every gram fish; Absolute ethyl alcohol with 2 times of above-mentioned 50% ethanol volumes washes residual fish tissue in the mortar again, and together changes in the centrifuge tube;

Step 3, homogenate were adopted ultrasonic-wave crushing 15～25 seconds; And under 4 ℃, 6000～8000 rev/mins centrifugal 10 minutes, collect supernatant;

Step 4, once with the washing of solid phase residue and 50% ethanol mixing, 50% ethanol washing consumption is that every gram fish is heavily used 1ml, again under 4 ℃, 6000～8000 rev/mins centrifugal 10 minutes, the collection supernatant;

Step 5, the supernatant in above-mentioned two steps is compiled, add the methylene chloride of 5 times of volumes of supernatant, repeatedly mixing, static layering then;

Step 6, absorb the water on upper strata with syringe, the centrifuge tube that will contain organic phase places fuming cupboard, 45 ℃ of water-baths, the organic phase of fully volatilizing; Fully dissolve with the PBS damping fluid that contains 1%BSA, and it is stand-by to be positioned over-20 ℃ of maintenances.

The present invention is according to the physicochemical property of steroid hormone, design steroid hormone extracting method efficiently, through experimental results show that repeatedly, the present invention can obtain high-quality steroid hormone and be used to research and analyse, and process radioimmunoassay method (RIA), chemoluminescence method and immune enzyme-linked method (ELISA) etc. determine the steroid hormone content in the young juvenile fish of multiple fish.Flow process of the present invention is simple, and is simple to operate, and the whole operation process is time-consuming few, and the result is reliable and stable, instrument that need not be special or expensive, and cost is low, and required reagent is few, and is nontoxic or low toxicity reagent, operating personnel is injured few.The present invention can be used for the extraction of the young juvenile fish steroid hormone of fish, is applicable to that also other aquatic biological grows and neuroendocrine regulation research, has extensive applicability.

Embodiment

Embodiment 1

1) get fresh turbot postlarva (total length 1.1cm, 0.10g), weighing is placed in the mortar and shreds;

2) in mortar, add 0.1ml 50% ethanol, after fully grinding homogenate is changed in the centrifuge tube; Wash residual fish tissue in the mortar with the 0.2ml absolute ethyl alcohol again, and together change in the centrifuge tube;

3) homogenate was adopted ultrasonic-wave crushing 15 seconds; And under 4 ℃, 6000 rev/mins centrifugal 10 minutes, collect supernatant;

4) with solid phase residue once with the washing of the 50% ethanol mixing of 0.1ml, again under 4 ℃, 6000 rev/mins centrifugal 10 minutes, collect supernatant;

5) supernatant in above-mentioned two steps is compiled, add the 2ml methylene chloride, repeatedly mixing, static layering then;

6) with the water on syringe absorption upper strata, the centrifuge tube that will contain organic phase places fuming cupboard, 45 ℃ of water-baths, the organic phase of fully volatilizing; Fully dissolve with the PBS that contains 1%BSA (pH 7.4) damping fluid 0.5ml, and it is stand-by to be positioned over-20 ℃ of maintenances.

The sample that this method is extracted adopt northern biotechnology research the testosterone radioimmunological kit, to measure its testosterone concentration be 0.301ng/g through putting the method for exempting from.

Embodiment 2

1) get freezing preservation the lefteye flounder juvenile fish (total length 5.8cm, 2.10g), weighing is placed in the mortar and shreds;

2) in mortar, add 2.1ml 50% ethanol, after fully grinding homogenate is changed in the centrifuge tube; Wash residual fish tissue in the mortar with the 4.2ml absolute ethyl alcohol again, and together change in the centrifuge tube;

3) homogenate was adopted ultrasonic-wave crushing 25 seconds; And under 4 ℃, 6000 rev/mins centrifugal 10 minutes, collect supernatant;

4) with solid phase residue once with the washing of the 50% ethanol mixing of 2.1ml, again under 4 ℃, 6000 rev/mins centrifugal 10 minutes, collect supernatant;

5) supernatant in above-mentioned two steps is compiled, add the 42ml methylene chloride, repeatedly mixing, static layering then;

6) with the water on syringe absorption upper strata, the centrifuge tube that will contain organic phase places fuming cupboard, 45 ℃ of water-baths, the organic phase of fully volatilizing; Fully dissolve with the PBS that contains 1%BSA (pH 7.4) damping fluid 1.0ml, and it is stand-by to be positioned over-20 ℃ of maintenances.

The sample that this method is extracted adopts the testosterone and the estradiol radioimmunological kit of Weifang, Shandong three dimensional diagnostic technology company limited, and to measure its testosterone concentration be 0.037ng/g through putting the method for exempting from, and its estradiol content is 25.12 μ g/g.

Embodiment 3

1) get fresh pomfret parr (total length 2.5cm, 0.11g), weighing is placed in the mortar and shreds;

2) in mortar, add 0.11ml 50% ethanol, after fully grinding homogenate is changed in the centrifuge tube; Wash residual fish tissue in the mortar with the 0.22ml absolute ethyl alcohol again, and together change in the centrifuge tube;

3) homogenate was adopted ultrasonic-wave crushing 25 seconds; And under 4 ℃, 6000 rev/mins centrifugal 10 minutes, collect supernatant;

4) with solid phase residue once with the washing of the 50% ethanol mixing of 0.11ml, again under 4 ℃, 6000 rev/mins centrifugal 10 minutes, collect supernatant;

5) supernatant in above-mentioned two steps is compiled, add the 2.2ml methylene chloride, repeatedly mixing, static layering then;

6) with the water on syringe absorption upper strata, the centrifuge tube that will contain organic phase places fuming cupboard, 45 ℃ of water-baths, the organic phase of fully volatilizing; Fully dissolve with the PBS that contains 1%BSA (pH 7.4) damping fluid 0.5ml, and it is stand-by to be positioned over-20 ℃ of maintenances.

The sample that this method is extracted adopt northern biotechnology research the estradiol radioimmunological kit, to measure its estradiol content be 47.17 μ g/g through putting the method for exempting from.

Claims (1)

1. young fast juvenile fish steroid hormone extracting method with young juvenile fish historrhexis, utilizes ethanol and methylene chloride that it is dissolved again, it is characterized in that concrete steps are as follows:

Step 1, get fresh or-20 ℃ of frozen young juvenile fish, shred after the weighing and place mortar;

Step 2, in mortar, adds 50% ethanol, fully after the grinding homogenate is changed in the centrifuge tube according to the ratio of heavy corresponding 1ml 50% ethanol of every gram fish; Absolute ethyl alcohol with 2 times of above-mentioned 50% ethanol volumes washes residual fish tissue in the mortar again, and together changes in the centrifuge tube;

Step 3, homogenate were adopted ultrasonic-wave crushing 15～25 seconds; And under 4 ℃, 6000～8000 rev/mins centrifugal 10 minutes, collect supernatant;

Step 4, once with the washing of solid phase residue and 50% ethanol mixing, 50% ethanol washing consumption is that every gram fish is heavily used 1ml, again under 4 ℃, 6000～8000 rev/mins centrifugal 10 minutes, the collection supernatant;

Step 5, the supernatant in above-mentioned two steps is compiled, add the methylene chloride of 5 times of volumes of supernatant, repeatedly mixing, static layering then;

Step 6, absorb the water on upper strata with syringe, the centrifuge tube that will contain organic phase places fuming cupboard, 45 ℃ of water-baths, the organic phase of fully volatilizing; Fully dissolve with the PBS damping fluid that contains 1%BSA, and it is stand-by to be positioned over-20 ℃ of maintenances.