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Solution and ultrasonic method for extracting earthworm coelomocytes

A cell liquid and cell technology, applied in biochemical equipment and methods, microorganisms, tissue culture, etc., can solve the problems of cumbersome operation, activity damage, and low survival rate, and achieve the effect of wide practicability, easy mastery, and simple operation

Inactive Publication Date: 2011-07-20
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although the number of coelomocytes is high, the survival rate is not high
Zhu Jiang et al. extracted the coelomocytes of earthworms by using fiber needles combined with ultrasonic stimulation, and optimized them. However, because live earthworms need to be acupunctured with fiber needles and thin tubes, the operation is difficult for some species of earthworms with small individuals. more cumbersome
The solution used to extract coelomocytes from earthworms generally uses EDTA or EGTA ammonia carboxyl complexing agent, or uses ethanol, etc. However, the number of coelomocytes extracted by these extracts is not large, and the activity is also damaged
The number and survival rate of earthworm coelomocytes extracted by the above methods can no longer meet the needs of cytotoxicology

Method used

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  • Solution and ultrasonic method for extracting earthworm coelomocytes
  • Solution and ultrasonic method for extracting earthworm coelomocytes
  • Solution and ultrasonic method for extracting earthworm coelomocytes

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1 Effects of different ultrasonic frequencies on the extraction of earthworm coelomocytes

[0027] The standard worm species for the international earthworm toxicity experiment——Eisenia fetida, a healthy adult worm with a mature annulus and a body weight of about 0.4 g, basically the same size, was selected. After 24 hours of bowel cleansing, the earthworms were repeatedly washed with PBS buffer solution at room temperature for 2-3 times, and the water on the surface of the earthworms was blotted with filter paper. Put the earthworms into 3mL of ice-cold extraction solution (60mM NaCl, 5%ethanol, 50mM guaicol glycerylether, 5mM EGTA) for extracting coelomocytes, and extract them quickly by ultrasonic method. The operating frequency of the ultrasonic instrument is 32KHz, 40KHz and 48KHz respectively, the power is 250W, extraction is performed twice, each ultrasonic time is 2s, and the temperature during ultrasonication is kept at 22°C. After ultrasonication, the...

Embodiment 2

[0032] Example 2 Effects of Different Ultrasonic Times on the Extraction of Earthworm Coelomocytes

[0033] The standard worm species for the international earthworm toxicity experiment——Eisenia fetida, a healthy adult worm with a mature annulus and a body weight of about 0.4 g, basically the same size, was selected. After 24 hours of bowel cleansing, the earthworms were repeatedly washed with PBS buffer solution at room temperature for 2-3 times, and the water on the surface of the earthworms was blotted with filter paper. Put the earthworms into 3 mL of ice-cold extraction solution for coelomocytes (60 mM NaCl, 5% ethanol, 50 mM guaicol glycerylether, 5 mM EGTA), and extract them quickly by ultrasonic method. The working frequency of the sonicator is 32KHz, the power is 250W, extraction is performed twice, each time of sonication is 1s, 2s and 3s, and the temperature during sonication is kept at 22°C. After ultrasonication, the earthworms were observed to vibrate continuous...

Embodiment 3

[0038] Example 3 Effects of Different Ultrasonic Temperatures on the Extraction of Earthworm Coelomocytes

[0039] The standard worm species for the international earthworm toxicity experiment——Eisenia fetida, a healthy adult worm with a mature annulus and a body weight of about 0.4 g, basically the same size, was selected. After 24 hours of bowel cleansing, the earthworms were repeatedly washed with PBS buffer solution at room temperature for 2-3 times, and the water on the surface of the earthworms was blotted with filter paper. Put the earthworms into 3 mL of ice-cold extraction solution for coelomocytes (60 mM NaCl, 5% ethanol, 50 mM guaicol glycerylether, 5 mM EGTA), and extract them quickly by ultrasonic method. The operating frequency of the ultrasonic instrument is 32KHz, the power is 250W, extraction is performed twice, and the ultrasonic time is 2s each time, and the ultrasonic temperature is 22°C and 37°C respectively. After ultrasonication, the earthworms were obs...

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Abstract

The invention discloses a solution and an ultrasonic method for extracting earthworm coelomocytes, belonging to the technical field of bioengineering. Each 3 mL of the solution for extracting the earthworm coelomocytes contains 60-70 mM of NaCl, 5-6% by weight of ethanol, 50-60 mM of guaicol glyceryl ether and 5-6mM of EGTA PBS buffer solution. The operation steps of the ultrasonic method are as follows: step 1, clearing the bowel of an earthworm, namely, using the PBS buffer solution for repeated cleaning; step 2, putting the earthworm into the cold solution for extracting the coelomocytes, and carrying out fast ultrasonic extraction; step 3, removing the earthworm, adding LBBS buffer solution for cleaning, centrifuging and removing supernate, wherein obtained precipitates are the coelomocytes; step 4, adding cell sap for incubation; and step 5, using trypan blue for coloring and microscopic examination, and calculating cell viability. The solution and the ultrasonic method extract a great many coelomocytes, realizes high cell viability, can satisfy the needs of apoptosis and a single cell gel electrophoresis experiment, and can be used for early diagnosis of soil pollution and function for early warning.

Description

technical field [0001] The invention relates to an extraction solution and an ultrasonic extraction method for bioengineering technology, in particular to a solution and an ultrasonic extraction method for extracting earthworm body coelomocytes. Background technique [0002] As an ancient organism, earthworms have existed in nature for 600 million years. They are one of the animal groups with the largest biomass in soil and are an important part of the soil system. Earthworms play important roles in the physical, chemical and biological processes of soil. They are widely distributed in the soil and play an important role in the degradation of organic matter, nutrient cycling and soil formation. At the same time, earthworms can accelerate the decomposition of carbon, affect the carbon allocation of the ecosystem, and directly or indirectly control the composition of plant communities and promote plant growth. Community succession, affecting terrestrial ecosystems. In the fo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06
Inventor 王晓蓉薛银刚姜锦林顾雪元吴可
Owner NANJING UNIV
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