CN101638634B - Solution and ultrasonic method for extracting earthworm coelomocytes - Google Patents
Solution and ultrasonic method for extracting earthworm coelomocytes Download PDFInfo
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- CN101638634B CN101638634B CN2009100342614A CN200910034261A CN101638634B CN 101638634 B CN101638634 B CN 101638634B CN 2009100342614 A CN2009100342614 A CN 2009100342614A CN 200910034261 A CN200910034261 A CN 200910034261A CN 101638634 B CN101638634 B CN 101638634B
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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
The present invention relates to a kind of extraction solution and ultrasonic extracting method that is used for biotechnology, extract the solution and the ultrasonic method of earthworm coelomocyte in particular.
Background technology
There have been 600,000,000 years in earthworm as a kind of ancient biology at nature, is one of animal monoid of biomass maximum in the soil, is integral part important in the soil system.Earthworm plays the part of important role in physics, chemistry and the bioprocess of soil.They are widely distributed in soil, in forming, organic matter degradation, Nutrient cycle and soil plays an important role, earthworm is by quickening the decomposition of carbon simultaneously, the carbon that influences the ecosystem distributes, the composition of controlling plant group directly or indirectly, promote Succession of Plant Community, influence TERRESTRIAL ECOSYSTEMS.In food chain, earthworm is the bridge that transmits pollutent between terrestrial organism and the soil organisms, is a kind of non-target Lu Sheng soil organisms of very important environment toxic substance.They are important species in the ecosystem and the Biological indicator who extensively is used as environmental pollution.Some earthworms (as Eisenia fetidai) occupy critical role in toxicological experiment, they are widely used in the environmental assessment of soil pollution, are the canonical biometrics of standard toxicological experiment.Initial purpose is in order to the genotoxic potential of assessing the chemical substance that newly enters environment and the toxicity of contaminated soil.Physiological metabolism of earthworm and vital movement have reflected the ecological functions of soil to a certain extent, therefore can estimate the soil ecology function and judge soil pollution situation and environmental quality by the employing worm biomarker, some biomarker also can be made objective judgement to the effect of combined pollution, and this is that the chemical detection method is difficult to accomplish usually.
The earthworm biomarker can be used as the effective tool of the accurate measurement of very early warning system or pollution hazard, the earthworm biomarker become the international research focus.International earthworm and ecological toxicology symposial (IWEE, International Workshop on Earthworm Ecotoxicology) 3 have been held so far, in two nearest progress and definite Future Development directions of all having set forth the earthworm biomarker.In in the past 5 years, research about the earthworm biomarker has obtained some significant results, such as: the variation of anti-oxidative defense system, the damage of DNA, metallothionein(MT) induce and NRRT analyzes etc., in recent years, the fast development of subject such as modern molecular biology, cytobiology, information biology and technology provides new method for ecological toxicologic study.By using Flow Cytometry, the molecular biosciences mark of earthworm is subjected to extensive concern as the early warning index of pollutent exposure and toxic effect on cell levels, and has become one of focus of domestic and international ecotoxicology research.Yet the biomarker of research earthworm on cell levels the most important thing is to extract the cell of earthworm earlier.
Find that by literature search up to the present, existing many methods are used to extract the coelomocyte of earthworm both at home and abroad to prior art.The early stage earthworm coelomocyte that obtains is can be easy to obtain coelomocyte and not infringement of pair cell by puncturing body cavity.1973, the method that human machinery such as Valembois and voltage stimulate induced coelomocyte to discharge from the earthworm body opening, and subsequently, Cooper proposes to utilize the pasteur pipet of point to obtain the coelomocyte of the positive earthworm in land.People such as Eyambe used alcohol as stimulator and added the mucus solute afterwards, and the cell quantity that obtains like this is the twice of original method approximately.Along with the raising to the coelomocyte requirement of experiment, extracting method is also in continuous improvement.People such as M.Hendawi use ultrasonic method to extract earthworm coelomocyte, the earthworm coelomocyte quantity that its quantity is obtained greater than Ethanol Method and voltage stimulus method.Yet though coelomocyte quantity is more, surviving rate is not high.Zhu Jiang etc. have extracted the coelomocyte of earthworm by adopting fiber needle in conjunction with the ultrasonic stimulation method, optimize, but owing to will adopt the fiber needle tubule to carry out acupuncture to the earthworm of live body, operation be but comparatively loaded down with trivial details for some individual less earthworm kinds.The solution of the earthworm extraction coelomocyte that adopted in the past generally adopts EDTA or EGTA ammonia carboxylic coordination agent, perhaps use ethanol etc., yet the coelomocyte quantity that these extracting solutions extract is few, and activity also is subjected to some damages.The quantity of the earthworm coelomocyte that above the whole bag of tricks extracted and survival rate can not satisfy the needs of cell toxicology.
Summary of the invention
1, the technical problem that will solve
The present invention mainly is at the deficiencies in the prior art, the solution and the ultrasonic method that extract earthworm coelomocyte are provided, by extraction solution of the present invention and ultrasonic method, it is higher to obtain more coelomocyte quantity and surviving rate, can satisfy the needs of cell toxicology experiments such as flow cytometry and single cell gel electrophoresis.
2, technical scheme
The present invention is achieved by the following technical solutions, mainly comprises the steps:
A kind of ultrasonic method that extracts earthworm coelomocyte:
Step (1), the earthworm gut purge is cleaned repeatedly with the PBS damping fluid.
Selecting earthworm for use is Eisenia foetida (Eisenia fetida), is international earthworm toxicity experimental standard earthworm kind.Test preposition cleaning soil and cultivate for some time in advance, select the about 0.4g of body weight then, big or small basically identical, the health with ripe endless belt becomes earthworm.After the earthworm gut purge 24 hours, at room temperature wash repeatedly earthworm 2-3 time, blot earthworm body surface moisture with filter paper with the PBS damping fluid.
Step (2) is put into the solution of ice-cold extraction coelomocyte with earthworm, extracts with ultrasonic method rapidly.
The solution that earthworm is put into the ice-cold extraction coelomocyte of 3mL extracts with ultrasonic method afterwards, and the operating frequency of ultrasonoscope is 32KHz, and power is 250W, extracts 2 times, and each ultrasonic time is 1s or 2s, and the temperature when keeping ultrasonic is at 22 ℃.
Step (3) is removed earthworm, adds the LBBS buffer solution for cleaning.After ultrasonic, observe earthworm and constantly tremble, make solution remove earthworm after the yellow muddiness of outlet gradually, and slightly draw yellow solution with pipettor and to ice-cold LBSS damping fluid, clean 2 times.4 ℃ of following centrifugal 10min, rotating speed is 1500 rev/mins, centrifugal removal supernatant liquor, gained be precipitated as coelomocyte.
LBSS damping fluid: 71.5mM NaCl, 4.8mM KCl, 1.1mM MgSO
47H
2O, 0.4mMKH
2PO
4, 0.3mM Na
2PO
4, 4.2mM NaHCO
3, regulate pH 7.3.After having joined solution, autoclaving is placed on 4 ℃ then and preserves down.
Step (4) adds enchylema and hatches, dyeing, microscopy in the gained precipitation.Adding a certain amount of RPMI-1640 enchylema breeds.Selected staining fluid is that concentration is 4% trypan blue dye liquor mother liquor, is diluted to 0.4% trypan blue dye liquor working fluid with the PBS damping fluid, and dyeing time is 3min, after trypan blue dyeing, can pass through microscope, directly mirror is counted down, realizes the more accurate quantitative analysis of pair cell survival rate.
A kind of solution that extracts earthworm coelomocyte wherein contains the PBS damping fluid of 60-70mM NaCl, weight content 5%-6%ethanol, 50-60mM guaicol glyceryl ether and 5-6mM EGTA in every 3mL.
The concentration of described PBS damping fluid is 0.01M, and the pH value is 7.2, and solution prepares 121 ℃ of sterilizations of back high pressure 15min, and cooling is placed on 4 ℃ and preserves standby down.
RPMI-1640 enchylema: dissolve 1640 dry powder with ultrapure water, use NaHCO
3Regulate pH value to 7.2.Adding penicillin and Streptomycin sulphate makes its concentration be respectively 100U/ml and 100ug/ml.Then in Bechtop, with the membrane filtration degerming in 0.22um aperture.Add 10% calf serum then, keep in Dark Place under 4 ℃.
3, beneficial effect
The present invention has optimized the condition of cell body cavity extracting solution and supersound extraction by disclosing a kind of solution and ultrasonic method that extracts earthworm coelomocyte, and compared with prior art, the present invention has the following advantages:
(1) fast and convenient.The solution of extraction coelomocyte of the present invention and ultrasonic method, it is bad so that the coelomocyte quantity of being carried is few, the activity is low to have solved cell extract, etc. problem, operate comparatively simple, grasp easily, and can obtain the coelomocyte of earthworm in a short period of time, can carry out early diagnosis to soil pollution by apoptosis and SCGE experiment, thereby realize the effect of early warning.
(2) the coelomocyte quantity of Ti Quing is more, and activity is stronger.The solution of extraction coelomocyte of the present invention and ultrasonic method, it is bad or extracting method is not good so that the coelomocyte quantity of being carried is few, active problem such as low to have solved cell extract, the quantity of the coelomocyte that is extracted is all more than other technologies, and the surviving rate of cell is all higher, reaches more than 90%.
(3) practicality is wider.The present invention is suitable for too to other Lumbricus kind.
Embodiment
Further specify the present invention by the following examples:
The influence that embodiment 1 different ultrasonic frequencies are extracted earthworm coelomocyte
Select international earthworm toxicity experimental standard earthworm kind---Eisenia foetida (Eisenia fetida) for use, the about 0.4g of body weight, big or small basically identical, the health with ripe endless belt becomes earthworm.After the earthworm gut purge 24 hours, at room temperature wash repeatedly earthworm 2-3 time, blot earthworm body surface moisture with filter paper with the PBS damping fluid.Earthworm is put among the solution 3mL (60mM NaCl, 5%ethanol, 50mM guaicol glycerylether, 5mM EGTA) of ice-cold extraction coelomocyte, extract with ultrasonic method rapidly.The operating frequency of ultrasonoscope is respectively at 32KHz, and 40KHz and 48KHz, power are 250W, extracts 2 times, and each ultrasonic time is 2s, and the temperature when keeping ultrasonic is at 22 ℃.After ultrasonic, observe earthworm and constantly tremble, remove earthworm after making solution engender yellow muddiness, and slightly draw yellow solution with pipettor and to ice-cold LBSS damping fluid, clean 2 times.4 ℃ of following centrifugal 10min, rotating speed is 1500 rev/mins, centrifugal removal supernatant liquor, gained be precipitated as coelomocyte.Adding a certain amount of RPMI-1640 enchylema in the gained precipitation breeds.Selected staining fluid is that concentration is 4% trypan blue dye liquor mother liquor, is diluted to 0.4% trypan blue dye liquor working fluid with the PBS damping fluid, and dyeing time is 3min, after trypan blue dyeing, can pass through microscope, directly mirror is counted down, realizes the more accurate quantitative analysis of pair cell survival rate.
Blood counting chamber and special-purpose cover glass are washed with ultrapure water after with 95% alcohol immersion, use the lens wiping paper wiped clean afterwards, and cover glass is overlying on the tally (the tally edge is earlier wetting, what cover glass pasted like this is better, and it is even that cell will distribute), with the abundant mixing of cell suspension, draw a little cell suspension with dropper and drop in the cover glass edge, make between cover glass and the tally to be full of cell suspension, attention is too full, in order to avoid overflow.Tally should be placed level, tilts, and leaves standstill 3min, allows cell sink, and microscopically is observed then, count plate four big lattice total cellular score, on the left of the line ball cell is only counted and top.Be calculated as follows: the big lattice total cellular score of cell count/ml=4/4 * 10000.The results are shown in Table 1.
The different ultrasonic frequencies of table 1 are to extracting the influence of earthworm coelomocyte
As known from Table 1, the earthworm coelomocyte that extracts during for 32KHz when ultrasonic operating frequency is relatively good.The earthworm coelomocyte per weight cell count of being extracted is 4.91 ± 0.05 (10
6Individual/g), surviving rate is 91.4%.
The influence that embodiment 2 different ultrasonic times extract earthworm coelomocyte
Select international earthworm toxicity experimental standard earthworm kind---Eisenia foetida (Eisenia fetida) for use, the about 0.4g of body weight, big or small basically identical, the health with ripe endless belt becomes earthworm.After the earthworm gut purge 24 hours, at room temperature wash repeatedly earthworm 2-3 time, blot earthworm body surface moisture with filter paper with the PBS damping fluid.Earthworm is put into the solution 3mL (60mM NaCl, 5%ethanol, 50mM guaicol glycerylether, 5mM EGTA) of ice-cold extraction coelomocyte, extract with ultrasonic method rapidly.The operating frequency of ultrasonoscope is at 32KHz, and power is 250W, extracts 2 times, and each ultrasonic time is 1s, 2s and 3s, and the temperature when keeping ultrasonic is at 22 ℃.After ultrasonic, observe earthworm and constantly tremble, make solution remove earthworm after the yellow muddiness of outlet gradually, and slightly draw yellow solution with pipettor and to ice-cold LBSS damping fluid, clean 2 times.4 ℃ of following centrifugal 10min, rotating speed is 1500 rev/mins, centrifugal removal supernatant liquor, gained be precipitated as coelomocyte.Adding a certain amount of RPMI-1640 enchylema in the gained precipitation breeds.Selected staining fluid is that concentration is 4% trypan blue dye liquor mother liquor, is diluted to 0.4% trypan blue dye liquor working fluid with the PBS damping fluid, and dyeing time is 3min, after trypan blue dyeing, can pass through microscope, directly mirror is counted down, realizes the more accurate quantitative analysis of pair cell survival rate.
Blood counting chamber and special-purpose cover glass are washed with ultrapure water after with 95% alcohol immersion, use the lens wiping paper wiped clean afterwards, and cover glass is overlying on the tally (the tally edge is earlier wetting, what cover glass pasted like this is better, and it is even that cell will distribute), with the abundant mixing of cell suspension, draw a little cell suspension with dropper and drop in the cover glass edge, make between cover glass and the tally to be full of cell suspension, attention is too full, in order to avoid overflow.Tally should be placed level, tilts, and leaves standstill 3min, allows cell sink, and microscopically is observed then, count plate four big lattice total cellular score, on the left of the line ball cell is only counted and top.Be calculated as follows: the big lattice total cellular score of cell count/ml=4/4 * 10000.The results are shown in Table 2.
The different ultrasonic times of table 2 are to extracting the influence of earthworm coelomocyte
As known from Table 2, the earthworm coelomocyte that extracts during for 2s when ultrasonic time is relatively good.The earthworm coelomocyte per weight cell count of being extracted is 4.87 ± 0.18 (10
6Individual/g), surviving rate is 91.8%.
The influence that embodiment 3 different ultrasonic temperature are extracted earthworm coelomocyte
Select international earthworm toxicity experimental standard earthworm kind---Eisenia foetida (Eisenia fetida) for use, the about 0.4g of body weight, big or small basically identical, the health with ripe endless belt becomes earthworm.After the earthworm gut purge 24 hours, at room temperature wash repeatedly earthworm 2-3 time, blot earthworm body surface moisture with filter paper with the PBS damping fluid.Earthworm is put into the solution 3mL (60mM NaCl, 5%ethanol, 50mM guaicol glycerylether, 5mM EGTA) of ice-cold extraction coelomocyte, extract with ultrasonic method rapidly.The operating frequency of ultrasonoscope is respectively at 32KHz, and power is 250W, extracts 2 times, and each ultrasonic time is 2s, and ultransonic temperature is respectively at 22 ℃ and 37 ℃.After ultrasonic, observe earthworm and constantly tremble, make solution remove earthworm after the yellow muddiness of outlet gradually, and slightly draw yellow solution with pipettor and to ice-cold LBSS damping fluid, clean 2 times.4 ℃ of following centrifugal 10min, rotating speed is 1500 rev/mins, centrifugal removal supernatant liquor, gained be precipitated as coelomocyte.Adding a certain amount of RPMI-1640 enchylema in the gained precipitation breeds.Selected staining fluid is that concentration is 4% trypan blue dye liquor mother liquor, is diluted to 0.4% trypan blue dye liquor working fluid with the PBS damping fluid, and dyeing time is 3min, after trypan blue dyeing, can pass through microscope, directly mirror is counted down, realizes the more accurate quantitative analysis of pair cell survival rate.
Blood counting chamber and special-purpose cover glass are washed with ultrapure water after with 95% alcohol immersion, use the lens wiping paper wiped clean afterwards, and cover glass is overlying on the tally (the tally edge is earlier wetting, what cover glass pasted like this is better, and it is even that cell will distribute), with the abundant mixing of cell suspension, draw a little cell suspension with dropper and drop in the cover glass edge, make between cover glass and the tally to be full of cell suspension, attention is too full, in order to avoid overflow.Tally should be placed level, tilts, and leaves standstill 3min, allows cell sink, and microscopically is observed then, count plate four big lattice total cellular score, on the left of the line ball cell is only counted and top.Be calculated as follows: the big lattice total cellular score of cell count/ml=4/4 * 10000.The results are shown in Table 3.
The different ultrasonic temperature of table 3 are to extracting the influence of earthworm coelomocyte
As known from Table 3, the earthworm coelomocyte that extracts when ultrasonic temperature is 22 ℃ is relatively good.The earthworm coelomocyte per weight cell count of being extracted is 4.88 ± 0.15 (10
6Individual/g), surviving rate is 91.8%.
Example 4 different cell extracts are to the influence of earthworm coelomocyte
Select international earthworm toxicity experimental standard earthworm kind---Eisenia foetida (Eisenia fetida) for use, the about 0.4g of body weight, big or small basically identical, the health with ripe endless belt becomes earthworm.After the earthworm gut purge 24 hours, at room temperature wash repeatedly earthworm 2-3 time, blot earthworm body surface moisture with filter paper with the PBS damping fluid.Earthworm is put into solution 3mL (two kinds of proportioning A, B and C, A:60mM NaCl, 5%ethanol, 50mM guaicol glyceryl ether, the 5mM EGTA of ice-cold extraction coelomocyte; B:65mM NaCl, 5.5%ethanol, 55mM guaicol glyceryl ether, 5.5mM EGTA C:70mM NaCl, 6%ethanol, 60mM guaicol glyceryl ether, 6mM EGTA) in, extract with ultrasonic method rapidly.The operating frequency of ultrasonoscope is 32KHz, and power is 250W, extracts 2 times, and each ultrasonic time is 2s, and the temperature when keeping ultrasonic is at 22 ℃.After ultrasonic, observe earthworm and constantly tremble, remove earthworm after making solution engender yellow muddiness, and slightly draw yellow solution with pipettor and to ice-cold LBSS damping fluid, clean 2 times.4 ℃ of following centrifugal 10min, rotating speed is 1500 rev/mins, centrifugal removal supernatant liquor, gained be precipitated as coelomocyte.Adding a certain amount of RPMI-1640 enchylema in the gained precipitation breeds.Selected staining fluid is that concentration is 4% trypan blue dye liquor mother liquor, is diluted to 0.4% trypan blue dye liquor working fluid with the PBS damping fluid, and dyeing time is 3min, after trypan blue dyeing, can pass through microscope, directly mirror is counted down, realizes the more accurate quantitative analysis of pair cell survival rate.
Blood counting chamber and special-purpose cover glass are washed with ultrapure water after with 95% alcohol immersion, use the lens wiping paper wiped clean afterwards, and cover glass is overlying on the tally (the tally edge is earlier wetting, what cover glass pasted like this is better, and it is even that cell will distribute), with the abundant mixing of cell suspension, draw a little cell suspension with dropper and drop in the cover glass edge, make between cover glass and the tally to be full of cell suspension, attention is too full, in order to avoid overflow.Tally should be placed level, tilts, and leaves standstill 3min, allows cell sink, and microscopically is observed then, count plate four big lattice total cellular score, on the left of the line ball cell is only counted and top.Be calculated as follows: the big lattice total cellular score of cell count/ml=4/4 * 10000.The results are shown in Table 4.
The different cell extracts of table 4 are to extracting the influence of earthworm coelomocyte
As known from Table 4, when ultrasonic operating frequency is 32KHz, extract temperature in the time of 22 ℃, different cell extract A, B, three groups of C are relatively good to the earthworm coelomocytes that extract in given range.The earthworm coelomocyte per weight cell count of being extracted is between 4.86-5.00 (10
6Individual/g), mean value 4.90 ± 0.11 (10
6Individual/g), surviving rate is between 90.5%-91.9, mean value 91.1% ± 0.02.
Claims (4)
1. ultrasonic method that extracts earthworm coelomocyte mainly comprises following several steps:
The first step with the earthworm gut purge, is cleaned repeatedly with the PBS damping fluid;
Second step, earthworm is put into the solution that is used to extract earthworm coelomocyte, this solution extracts with ultrasonic rapidly in the PBS damping fluid that every 3mL wherein contains 60-70mM NaCl, weight content 5%-6%ethanol, 50-60mM guaicol glycerylether and 5-6mM EGTA;
The 3rd step, remove earthworm, add the LBSS buffer solution for cleaning, centrifugal, remove supernatant, gained is precipitated as coelomocyte;
In the 4th step, add enchylema and hatch;
In the 5th step, trypan blue dyeing, microscopy calculate cell survival rate.
2. a kind of ultrasonic method that extracts earthworm coelomocyte according to claim 1, it is characterized in that ultransonic extraction conditions in second step: the operating frequency of ultrasonic apparatus is 32KHz, and power is 250W, extracts 2 times, each ultrasonic time is 1s or 2s, controls ultransonic temperature at 22 ℃.
3. a kind of ultrasonic method that extracts earthworm coelomocyte according to claim 2, it is characterized in that the 3rd the step under 4 ℃ of conditions centrifugal 10min, rotating speed is 1500 rev/mins, centrifugal removal supernatant liquor, gained be precipitated as coelomocyte, add a certain amount of RPMI-1640 enchylema then and hatch.
4. according to each described a kind of ultrasonic method that extracts earthworm coelomocyte in the claim 1～3, the concentration that it is characterized in that described trypan blue dye liquor mother liquor of the 5th step is 4%, be diluted to 0.4% trypan blue dye liquor working fluid with the PBS damping fluid, dyeing time is 3min, after trypan blue dyeing, can pass through microscope, directly mirror is counted down, realizes the more accurate quantitative analysis of pair cell survival rate.
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