CN101982075B - Biological model of steroids hormone pollutant in foods and feeds established by utilizing Caenorhadits elegans and method thereof - Google Patents
Biological model of steroids hormone pollutant in foods and feeds established by utilizing Caenorhadits elegans and method thereof Download PDFInfo
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Abstract
The invention relates to a biological model of steroids hormone pollutant in foods and feeds established by utilizing Caenorhadits elegans and a method thereof, belonging to the field of a safety detection technique of foods and feeds. The biological model of the steroids hormone pollutant in the foods and the feeds is established by utilizing the Caenorhadits elegans. The method of the invention is characterized in that the Caenorhadits elegans is utilized to establish a biological model of steroids hormone pure products; the Caenorhadits elegans is utilized to establish the biological model of steroids hormone in the foods; and the Caenorhadits elegans is utilized to establish a biological model of steroids hormone in the feeds. The invention has the beneficial effects of important significance of the research of the steroids hormone to influence on human health, and short period, high efficiency, conciseness and convenience of the experiment course, less requirements on instruments, low cost and great application value. Moreover, the period of the Caenorhadits elegans from the first stage of larvae to the stage of oviposition and the generation time are detected to identify the precocity phenomenon of organisms under the influence of the steroids hormone by exposing the steroids hormone of the Caenorhadits elegans in the first stage of the larvae.
Description
Technical field
The invention belongs to technical fields such as food safety detection, feed safety detection.
Background technology
(Caenorhabditis elegans is a kind of nematode that can independently survive C.elegans) to Caenorhabditis elegans, and its individuality is little; Adult only 1.5mm is long, is hermaphroditism, and male only accounts for 0.2% of colony; But self-fertilization or two sexual reproduction are 3.5 days 20 ℃ of following average history of life, and average fertility is 300-350; But if with the male worm mating, can produce the nearly offspring more than 1400.The genome size of beautiful nematode is 97Mb; Be distributed in 6 chromosomes; Prediction has 19717 of encoding genes, genome sequence 40% and human homology, and this just means it and the more total similar cells of more high organism and molecular structure and control access; It is first genosome fully by the many cells eucaryote of sequencing, and each somatic developmental state all studies comparatively clearly, the rule of this cell line is almost constant between each individuals; Its genetic background is clear, individual configurations is simple, the history of life is short; And sample is cheap, control easily in the laboratory.Therefore be used widely in fields such as the emergency reaction of old and feeble and life-span, human genetic disease, animal, environmentals.For example: the method (publication number: CN 101451983A) etc. of measuring the generation toxicity of medicine and personal care articles with beautiful nematode.
Steroid hormone is a compounds of numerous environmental hormone class materials, is characterized in having very high stability, and human endocrine is had interference effect.In recent years, to show that harm that steroid hormone causes wildlife mainly shows as reproductive organs, reproduction function and reproductive behavio(u)r unusual about scholar's research.For example: through PCB-60 ,-104 ,-190 and oestrogenic hormone 17 beta estradiols the experimental study of zebra fish is shown that these hormone substances can increase fish embryo and young lethality greatly; The amphipod of long term exposure in 10 μ g/L nonyl phenol environment, its population density and growth rate obviously reduce; The male flatfish that lives in the Britain bay is owing to receive the influence of environmental hormone, and its blood vitellin concentration has trend of rising, and reproductive behavior is unusual; The crocodile young in the Fla. polluted lake, owing to receive the influence of environmental hormone, the adjusting of sex hormone and the concentration of thyroid hormone change to some extent in their bodies.In animal body residual of steroid hormone gets into human body through food chain, and people's normal physiological function is got muddled, and child's sexual maturity is accelerated, and influences normal growth and growth.
In the food steroid hormone be mainly derived from beginning of production animals and plants self steroid hormone, make the steroid hormone in the medicament as the exogenous steroid hormone of animal feed additive and the excessive use of plant growth regulator and in order to prevent, to treat the animals and plants disease and promoting output to animals and plants, cause that the steroid hormone level is remarkable ascendant trend in the food.Domestic milk powder is urged child's sex premature incident, makes country pay much attention to local governments at various levels.Therefore utilize the beautiful nematode of many cells eucaryote to set up that steroid hormone pollutant safety living model is very important in food and the feed, for steroid hormone pollutant detection in food and the feed provides new method.
The domestic and international at present research method to steroid hormone in food and the feed mainly contains chemical method and biological methods such as high performance liquid chromatography, radio immunoassay, AAS, TLC, gas chromatography-mass spectrum (GC-MS) method; These methods all are to study steroid hormone from quantitative angle; And cost is high, the cycle is long, and is also higher to experimenter's specification requirement.
Summary of the invention
The objective of the invention is: the method that provides the beautiful nematode living model of a kind of usefulness that the radical row of steroidal class is detected; Utilize the beautiful nematode of many cells eucaryote to set up steroid hormone pollutant safety living model in food and the feed, for the mankind's food-safety problem provides theoretical, technical foundation and novel detection method.
Technical scheme of the present invention is following:
Set up steroid hormone pollutant living model in food and the feed with beautiful nematode.
Method of the present invention is:
One, utilize beautiful nematode living model that the pure article of steroid hormone are carried out the method that safety hazards detects:
1, obtain worm liquid of the synchronized beautiful nematode larval first phase (L1 phase): just the beautiful nematode hermaphroditism adult in the egg-laying period washes the centrifugal 1min of 1500rpm, supernatant discarded from the NGM culture plate with the M9 buffer solution.As above method will contain beautiful nematode after once with the M9 buffer solution for cleaning again M9 buffer solution constant volume in container, and add the equal-volume lysate, the centrifugal 3min of 1500rpm behind the mixing 3-5min that turns upside down, supernatant discarded is collected bottom precipitation.Deposition is used aseptic S-Medium instead and is cleaned 2-3 time repeatedly and reach neutrality to pH value, and at this moment beautiful nematode polypide is by cracking and worm's ovum is still intact.The aseptic S-Medium solution that will contain beautiful nematode worm's ovum is put in (16 ℃-25 ℃) cultivation 8-12h in the biochemical incubator.Owing to there is not food, egg hatch can all stop at the beautiful nematode larval first phase (L1 phase) later on, and the centrifugal 3min of 1500rpm collects larva, and is subsequent use.
2, the pure article of steroid hormone being dissolved in concentration expressed in percentage by volume is in 1% the dimethyl sulfoxide (DMSO).
3, the beautiful nematode larval first phase (L1 phase) worm liquid is exposed in the pure article of steroid hormone:
Beautiful nematode adopts the liquid exposure method to expose; Two groups of samples are set; First group is exposed group: with aseptic Tissue Culture Plate; Every hole adds the worm liquid that is in the beautiful nematode larval first phase (L1 phase) and E.coli OP50 and liquid NGM culture fluid and the pure article of steroid hormone that 1 collection step obtains, and mixing, and the final concentration of steroid hormone is 0.1ng/ml-20ng/ml; Second group is the blank group: with aseptic Tissue Culture Plate, every hole adds the worm liquid that is in the beautiful nematode larval first phase (L1 phase) and E.coli OP50 and the liquid NGM culture fluid that 1 collection step obtains.These two groups of samples are put into liquid-transfering gun sample is taken out respectively after biochemical incubator (16 ℃-25 ℃) expose to be cultivated 6h, place centrifuge tube.
4, in the centrifuge tube of two groups of samples, add the M9 buffer solution respectively, leave standstill 10min after shaking up, remove supernatant, add the M9 buffer solution again, leave standstill 10min after shaking up, remove supernatant again, add the M9 buffer solution again and shake up.Two groups of samples taking-ups are coated respectively on the NGM solid culture medium that contains E.coli OP50; In biochemical incubator (16 ℃-25 ℃), cultivate up to nematode long till the length that can choose; Nematode is chosen, put into respectively on the new NGM solid culture medium that contains E.coli OP50, cultivate in the biochemical incubator (16 ℃-25 ℃); Every other day nematode is chosen to new containing on the E.coli OP50 NGM solid culture medium, finished up to the egg-laying period.The nematode of losing, should from statistical data, get rid of because of climbing to culture dish wall dead nematode and " worm bag " (worm bag).Adopt stereomicroscope with imaging software beautiful nematode is recorded a video, carry out the mensuration of following index:
(1) steroid hormone is to the influence of beautiful nematode genital system:
A, from the beautiful nematode larval first phase (L1 phase) needed time when beginning to lay eggs.
B, offspring's number: offspring's number is all ovum numbers in whole egg-laying period.
The time interval that C, generation time: P0 is laid eggs and laid eggs for adult to its F1 for adult.
(2) steroid hormone is to the influence of beautiful nematode locomitivity:
A, head hunting frequency: will grow for the nematode of adult and choose in the solution, and, write down the number of times (swinging as 1 head) of nematode head swing in 1min when the health bending reaches long half of body through the recovery of 1min.
B, health corner frequency: will grow for the nematode of adult and choose on the NGM medium that does not have an OP50, and note the crooked number of times of health in 20 s.Suppose that along the direction of antlia be the y axle, health is defined as 1 health bending along 1 change on the corresponding x direction of principal axis in the nematode crawling process so.
(3) steroid hormone influence that beautiful nematode is grown:
A, life-span: with initial 0 day of nematode life of line eggs hatching beginning to be designated as, to stop with getting the terminal light reactionless nematode life that is designated as of polypide of visiting of worm device.The nematode lifetime is designated as nematode from the initial time that stops to life of life.
B, the long mensuration of body: the nematode health is stiff, and the body when not having bending is long.
5, repeat 1,2,3,4 steps, and the open-assembly time of beautiful nematode is revised as 12h, 18h, 24h respectively.Obtain to expose 12h, steroid hormone is to the result that influences of beautiful nematode each item physical signs; Expose 18h, steroid hormone is to the result that influences of beautiful nematode each item physical signs; Expose 24h, steroid hormone is to the result that influences of beautiful nematode each item physical signs.
6, adopt software Origin7.5 that data are carried out significance analysis; Have significant difference if be exposed to the physical signs of the beautiful nematode in the steroid hormone with the physical signs contrast that is not exposed to the beautiful nematode in the steroid hormone, explain that then steroid hormone has safety hazards to beautiful nematode; If there is not significant difference, explain that then steroid hormone does not have safety hazards to beautiful nematode.
Two, utilize beautiful nematode living model that steroid hormone pollutant in the food is carried out the method that safety hazards detects:
1, the worm liquid of the beautiful nematode larval first phase of synchronization (L1 phase) of the acquisition of the step 1 in the repeat techniques scheme one.
2, fresh experiment is cleaned up with food,,, get and place homogenizer to be twisted into the mud shape in right amount with filter paper filter solid carbon dioxide branch with stainless steel scalpel section; Place centrifuge tube, add concentration expressed in percentage by volume and be in 1% the dimethyl sulfoxide (DMSO), stir with glass rod; Vortex oscillation 1min places 55 ℃ of water-baths to be incubated 20min, takes out vortex oscillation 20min; With the centrifugal 20min of 3000rpm, the sucking-off supernatant is the steroid hormone extract, and to be concentrated to concentration be more than 80%.
3, the beautiful nematode larval first phase (L1 phase) worm liquid is exposed in the steroid hormone extract:
Beautiful nematode adopts the liquid exposure method to expose; Two groups of samples are set; First group is exposed group: with aseptic Tissue Culture Plate; Every hole adds the worm liquid that is in the beautiful nematode larval first phase (L1 phase) and E.coli OP50 and liquid NGM culture fluid and the steroid hormone extract that obtains, and mixing; Second group is the blank group: with aseptic Tissue Culture Plate, every hole adds the worm liquid that is in the beautiful nematode larval first phase (L1 phase) and E.coli OP50 and the liquid NGM culture fluid that obtains.These two groups of samples are put into liquid-transfering gun sample is taken out respectively after biochemical incubator (16 ℃-25 ℃) expose to be cultivated 6h, place centrifuge tube.
4, the step 4,5,6 in the repeat techniques scheme one.
Three, utilize beautiful nematode living model that steroid hormone pollutant in the feed is carried out the method that safety hazards detects:
1, the step 1 in the repeat techniques scheme one obtains worm liquid of the synchronized beautiful nematode larval first phase (L1 phase).
2, get feed in tool plug centrifuge tube, add chloroform, shaken is also carried out natural evaporation.Add 100% alcohol dissolving steroidal compounds (concentrating 1000 times) again.Take out supernatant solution, be the steroid hormone extract.The liquid that is extracted is put as in the fume hood that it is air-dry, and using concentration expressed in percentage by volume is 1% dmso solution residue, and to be concentrated to concentration be more than 80%.
3, the beautiful nematode larval first phase (L1 phase) worm liquid is exposed in the steroid hormone extract:
Beautiful nematode adopts the liquid exposure method to expose; Two groups of samples are set; First group is exposed group: with aseptic Tissue Culture Plate; Every hole adds the worm liquid that is in the beautiful nematode larval first phase (L1 phase) and E.coli OP50 and liquid NGM culture fluid and the steroid hormone extract that obtains, and mixing; Second group is the blank group: with aseptic Tissue Culture Plate, every hole adds the worm liquid that is in the beautiful nematode larval first phase (L1 phase) and E.coli OP50 and the liquid NGM culture fluid that obtains.These two groups of samples are put into liquid-transfering gun sample is taken out respectively after biochemical incubator (16 ℃-25 ℃) expose to be cultivated 6h, place centrifuge tube.
4, the step 4,5,6 in the repeat techniques scheme one.
The invention has the beneficial effects as follows:
1, beautiful nematode is that first genosome is fully by the many cells eucaryote of sequencing; 19717 of encoding genes are arranged; About 35% gene has homologue in human body; Can be used as research people's various vital movements, the outstanding model biology of biological phenomena, susceptibility is high, and the influence to human health is significant to the research steroid hormone.
2, the experimentation cycle short, efficient, succinct, convenient, to instrument requirement few (stereomicroscope), cost is low, using value is big; The observation of experimental result is directly perceived; Be exposed to the physical signs of the beautiful nematode in the steroid hormone and have significant difference with the physical signs that is not exposed to the beautiful nematode in the steroid hormone, never ipsilateral has reflected that really steroid hormone is to the beautiful nematode genital system and the influence of growing.
3, utilized the experiment of beautiful nematode assess sample toxicity is the beautiful nematode that is in the larva fourth phase (L4 phase) to be carried out sample expose in the past; This experiment is that the beautiful nematode that is in the larva first phase (L1 phase) is carried out the exposure of steroid hormone; Measure beautiful nematode and reach generation time during this period of time to what begin to lay eggs from the larva first phase (L1 phase), identify the biological precocious phenomenon under the steroid hormone influence; Measure offspring's number of beautiful nematode, identify the harm of steroid hormone organism reproduction function; Measure other effects on physiological indexes of beautiful nematode, identify the influence that steroid hormone grows to beautiful nematode.
Embodiment
Embodiment 1: set up the pure article living model of estradiol with beautiful nematode
1, obtain worm liquid of the synchronized beautiful nematode larval first phase (L1 phase):
The beautiful nematode hermaphroditism adult in the egg-laying period is just washed the centrifugal 1min of 1500rpm, supernatant discarded from 90mm NGM culture plate with the M9 buffer solution.As above method is settled to 500 μ l in 1.5ml EP pipe with the M9 buffer solution that the M9 buffer solution for cleaning will contain beautiful nematode after once again.Add lysate 500 μ l, the centrifugal 3min of 1500rpm behind the mixing 3-5min that turns upside down, supernatant discarded is collected bottom precipitation.Deposition is used aseptic S-Medium instead and is cleaned 2-3 time repeatedly to pH value to 7.0, and beautiful nematode polypide is by cracking and worm's ovum is still intact at this moment.The aseptic S-Medium solution that will contain beautiful nematode worm's ovum is put in 20 ℃ and cultivates 8-12h.Owing to there is not food, egg hatch can all stop at the larva first phase later on, and the centrifugal 3min of 1500rpm collects larva.
2, the pure article of estradiol being dissolved in concentration expressed in percentage by volume is in 1% the dimethyl sulfoxide (DMSO).
3, the beautiful nematode larval first phase (L1 phase) worm liquid is exposed in the estradiol:
Beautiful nematode adopts the liquid exposure method to expose; Two groups of samples are set, and first group is exposed group: get 96 orifice plates, every hole adds the worm liquid 100 μ l that are in the larva first phase (L1 phase) that 1 collection step obtains; E.coli OP50 is to OD600=0.250 μ l; Liquid NGM culture fluid 49 μ l, estradiol solution 1 μ l, and mixing.Larva is exposed in the estradiol of four variable concentrations; The estradiol final concentration is respectively: 0.1ng/ml, 1.0ng/ml, 10ng/ml, 20ng/ml be totally four samples; Each sample establish four parallel; With liquid-transfering gun sample is taken out respectively after 6h is cultivated in 20 ℃ of exposures, four parallel inserting in the same 15ml centrifuge tube of each sample obtain the sample of four variable concentrations.Second group is the blank group: get 96 orifice plates, every hole adds the worm liquid that is in the larva first phase (L1 phase) the 100 μ l E.coli OP50 of 1 collection step acquisition to OD600=0.250 μ l, liquid NGM culture fluid 50 μ l.If four parallel, after cultivating 6h, 20 ℃ of exposures with liquid-transfering gun four parallel sample are taken out four parallel inserting in the same 15ml centrifuge tube respectively.
4, in centrifuge tube, add 4ml M9 buffer solution respectively, leave standstill 10min after shaking up, remove supernatant, add 4ml M9 buffer solution again, leave standstill 10min after shaking up, remove supernatant again, add an amount of M9 buffer solution again and shake up.Taking out 100 μ l in each sample coats on the NGM solid culture medium that contains 150 μ l E.coli OP50; 20 ℃ of cultivations are long till the length that can choose up to nematode; Nematode is chosen, is put on the new NGM solid culture medium that contains 150 μ l E.coli OP50, this moment each sample establish again four parallel; Choose during each is parallel into ten nematodes; 20 ℃ of cultivations are every other day chosen nematode to new containing on the 150 μ l E.coli OP50 NGM solid culture mediums, finish up to the egg-laying period.The nematode of losing, should from statistical data, get rid of because of climbing to culture dish wall dead nematode and " worm bag " (worm bag).Adopt stereomicroscope with imaging software beautiful nematode is carried out the video recording of 3min, carry out the mensuration of following index:
(1) steroid hormone is to the influence of beautiful nematode genital system:
A, from the beautiful nematode larval first phase (L1 phase) larva needed time when beginning to lay eggs.
B, offspring's number: i.e. all ovum numbers in whole egg-laying period.
The time interval that C, generation time: P0 is laid eggs and laid eggs for adult to its F1 for adult.
(2) steroid hormone is to the influence of beautiful nematode locomitivity:
A, head hunting frequency: will grow for the nematode of adult and choose in the solution, and, write down the number of times (swinging as 1 head) of nematode head swing in 1min when the health bending reaches long half of body through the recovery of 1min.
B, health corner frequency: will grow for the nematode of adult and choose on the NGM medium that does not have an OP50, and note the crooked number of times of health in the 20s.Suppose that along the direction of antlia be the y axle, health is defined as 1 health bending along 1 change on the corresponding x direction of principal axis in the nematode crawling process so.
(3) steroid hormone influence that beautiful nematode is grown:
A, life-span: with initial 0 day of nematode life of line eggs hatching beginning to be designated as, to stop with getting the terminal light reactionless nematode life that is designated as of polypide of visiting of worm device.The nematode lifetime is designated as nematode from the initial time that stops to life of life.
B, the long mensuration of body: the nematode health is stiff, and the body when not having bending is long.
5, repeat 1,2,3,4 steps, and the open-assembly time of beautiful nematode is revised as 12h, 18h, 24h respectively.Obtain to expose 12h, steroid hormone is to the result that influences of beautiful nematode each item physical signs; Expose 18h, steroid hormone is to the result that influences of beautiful nematode each item physical signs; Expose 24h, steroid hormone is to the result that influences of beautiful nematode each item physical signs.
6, adopt software Origin7.5 that data are carried out significance analysis; Have significant difference if be exposed to the physical signs of the beautiful nematode in the steroid hormone with the physical signs contrast that is not exposed to the beautiful nematode in the steroid hormone, explain that then steroid hormone has safety hazards to beautiful nematode; If there is not significant difference, explain that then steroid hormone does not have safety hazards to beautiful nematode.
Embodiment 2: set up steroid hormone living model in the pork with beautiful nematode
1, obtain worm liquid of the synchronized beautiful nematode larval first phase (L1 phase):
The beautiful nematode hermaphroditism adult in the egg-laying period is just washed the centrifugal 1min of 1500rpm, supernatant discarded from 90mm NGM wall panel with the M9 buffer solution.As above method is settled to 500 μ l in 1.5ml EP pipe with the M9 buffer solution that the M9 buffer solution for cleaning will contain beautiful nematode after once again.Add lysate 500 μ l, the centrifugal 3min of 1500rpm behind the mixing 3-5min that turns upside down, supernatant discarded is collected bottom precipitation.Deposition is used aseptic S-Medium instead and is cleaned 2-3 time repeatedly to pH value to 7.0, and beautiful nematode polypide is by cracking and worm's ovum is still intact at this moment.The aseptic S-Medium solution that will contain beautiful nematode worm's ovum is put in 20 ℃ and cultivates 8-12h.Owing to there is not food, egg hatch can all stop at the larva first phase (L1 phase) later on, and the centrifugal 3min of 1500rpm collects larva.
2, the processing of meat: fresh experiment is cleaned up with meat, cut edible muscle parts, with filter paper filter solid carbon dioxide branch with the stainless steel scalpel; Get and in homogenizer, be twisted into the meat gruel shape in right amount, take by weighing 10g and place centrifuge tube, add concentration expressed in percentage by volume and be in 1% the dimethyl sulfoxide (DMSO); Stir with glass rod, vortex oscillation 1min places 55 ℃ of water-baths to be incubated 20min; Take out vortex oscillation 20min; With the centrifugal 20min of 3000rpm, the sucking-off supernatant is the steroid hormone extract, and is concentrated to suitable concentration.
3, the beautiful nematode larval first phase (L1 phase) worm liquid is exposed in the steroid hormone extract:
Beautiful nematode adopts the liquid exposure method to expose; Two groups of samples are set, and first group is exposed group: get 96 orifice plates, every hole adds the worm liquid 100 μ l that are in the larva first phase (L1 phase) that 1 collection step obtains; E.coli OP50 is to OD600=0.250 μ l; Liquid NGM culture fluid 49 μ l, steroid hormone extract 1 μ l, and mixing.Establish altogether four parallel, 20 ℃ are taken out parallel sample with liquid-transfering gun four parallel inserting in the same 15ml centrifuge tube respectively after expose cultivating 6h.Second group is the blank group: get 96 orifice plates, every hole adds the worm liquid 100 μ l that are in the larva first phase (L1 phase) that 1 collection step obtains, and E.coli OP50 is to OD600=0.2 50 μ l, liquid NGM culture fluid 50 μ l.If four parallel, after cultivating 6h, 20 ℃ of exposures with liquid-transfering gun four parallel sample are taken out four parallel inserting in the same 15ml centrifuge tube respectively.4, in centrifuge tube, add 4ml M9 buffer solution respectively, leave standstill 10min after shaking up, remove supernatant, add 4ml M9 buffer solution again, leave standstill 10min after shaking up, remove supernatant again, add an amount of M9 buffer solution again and shake up.Taking out 100 μ l in each sample coats on the NGM solid culture medium that contains 150 μ l E.coli OP50; 20 ℃ of cultivations are long till the length that can choose up to nematode; Nematode is chosen, is put on the new NGM solid culture medium that contains 150 μ l E.coli OP50, this moment each sample establish again four parallel; Put into ten nematodes during each is parallel; 20 ℃ of cultivations are every other day chosen nematode to new containing on the 150 μ l E.coli OP50 NGM solid culture mediums, finish up to the egg-laying period.The nematode of losing, should from statistical data, get rid of because of climbing to culture dish wall dead nematode and " worm bag " (worm bag).Adopt stereomicroscope with imaging software beautiful nematode is carried out the video recording of 3min, carry out the mensuration of following index:
(1) steroid hormone is to the influence of beautiful nematode genital system:
A, from the beautiful nematode larval first phase (L1 phase) needed time when beginning to lay eggs.
B, offspring's number: i.e. all ovum numbers in whole egg-laying period.
The time interval that C, generation time: P0 is laid eggs and laid eggs for adult to its F1 for adult.
(2) steroid hormone is to the influence of beautiful nematode locomitivity:
A, head hunting frequency: will grow for the nematode of adult and choose in the solution, and, write down the number of times (swinging as 1 head) of nematode head swing in 1min when the health bending reaches long half of body through the recovery of 1min.
B, health corner frequency: will grow for the nematode of adult and choose on the NGM medium that does not have an OP50, and note the crooked number of times of health in 20 s.Suppose that along the direction of antlia be the y axle, health is defined as 1 health bending along 1 change on the corresponding x direction of principal axis in the nematode crawling process so.
(3) steroid hormone influence that beautiful nematode is grown:
A, life-span: with initial 0 day of nematode life of line eggs hatching beginning to be designated as, to stop with getting the terminal light reactionless nematode life that is designated as of polypide of visiting of worm device.The nematode lifetime is designated as nematode from the initial time that stops to life of life.
B, the long mensuration of body: the nematode health is stiff, and the body when not having bending is long.
5, repeat 1,2,3,4 steps, and the open-assembly time of beautiful nematode is revised as 12h, 18h, 24h respectively.Obtain to expose 12h, steroid hormone is to the result that influences of beautiful nematode each item physical signs; Expose 18h, steroid hormone is to the result that influences of beautiful nematode each item physical signs; Expose 24h, steroid hormone is to the result that influences of beautiful nematode each item physical signs.
6, adopt software Origin7.5 that data are carried out significance analysis; Have significant difference if be exposed to the physical signs of the beautiful nematode in the steroid hormone with the physical signs contrast that is not exposed to the beautiful nematode in the steroid hormone, explain that then steroid hormone has safety hazards to beautiful nematode; If there is not significant difference, explain that then steroid hormone does not have safety hazards to beautiful nematode.
Embodiment 3: set up steroid hormone living model in the milk powder with beautiful nematode
1, obtain worm liquid of the synchronized beautiful nematode larval first phase (L1 phase):
The beautiful nematode hermaphroditism adult in the egg-laying period is just washed the centrifugal 1min of 1500rpm, supernatant discarded from the 90mmNGM culture plate with the M9 buffer solution.As above method is settled to 500 μ l in 1.5ml EP pipe with the M9 buffer solution that the M9 buffer solution for cleaning will contain beautiful nematode after once again.Add lysate 500 μ l, the centrifugal 3min of 1500rpm behind the mixing 3-5min that turns upside down, supernatant discarded is collected bottom precipitation.Deposition is used aseptic S-Medium instead and is cleaned 2-3 time repeatedly to pH value to 7.0, and beautiful nematode polypide is by cracking and worm's ovum is still intact at this moment.The aseptic S-Medium solution that will contain beautiful nematode worm's ovum is put in 20 ℃ and cultivates 8-12h.Owing to there is not food, egg hatch can all stop at the larva first phase (L1 phase) later on, and the centrifugal 3min of 1500rpm collects larva.
2, accurately take by weighing 5g (being accurate to 0.1g) milk powder in 50mL tool plug centrifuge tube, add the 50mL chloroform, shaken is also carried out natural evaporation.Add 1mL 100% alcohol dissolving steroidal compounds (concentrating 1000 times) again.Take out supernatant solution, be the steroid hormone extract.The liquid that is extracted is put as in the fume hood that it is air-dry, and the use concentration expressed in percentage by volume is 1% dmso solution residue, and is concentrated to suitable concentration.
3, the beautiful nematode larval first phase (L1 phase) worm liquid is exposed in the steroid hormone extract:
Beautiful nematode adopts the liquid exposure method to expose, and two groups of samples are set, and first group is exposed group: get 96 orifice plates; Every hole adds the worm liquid 100 μ l that are in the larva first phase (L1 phase) that 1 collection step obtains, and E.coli OP50 is to OD600=0.250 μ l, liquid NGM culture fluid 49 μ l; Steroid hormone extract 1 μ l; Establish altogether four parallel, 20 ℃ are taken out sample with liquid-transfering gun four parallel inserting in the same 15ml centrifuge tube respectively after expose cultivating 6h.Second group is the blank group: get 96 orifice plates, every hole adds the worm liquid 100 μ l that are in the larva first phase (L1 phase) that 1 collection step obtains, and E.coli OP50 is to OD600=0.2 50 μ l, liquid NGM culture fluid 50 μ l.If four parallel, after cultivating 6h, 20 ℃ of exposures with liquid-transfering gun four parallel sample are taken out four parallel inserting in the same 15ml centrifuge tube respectively.
4, in centrifuge tube, add 4ml M9 buffer solution respectively, leave standstill 10min after shaking up, remove supernatant, add 4ml M9 buffer solution again, leave standstill 10min after shaking up, remove supernatant again, add an amount of M9 buffer solution again and shake up.Taking out 100 μ l in each sample coats on the NGM solid culture medium that contains 150 μ l E.coli OP50; 20 ℃ of cultivations are long till the length that can choose up to nematode; Nematode is chosen, is put on the new NGM solid culture medium that contains 150 μ l E.coli OP50, this moment each sample establish again four parallel; Put into ten nematodes during each is parallel; 20 ℃ of cultivations are every other day chosen nematode to new containing on the 150 μ l E.coli OP50 NGM solid culture mediums, finish up to the egg-laying period.The nematode of losing, should from statistical data, get rid of because of climbing to culture dish wall dead nematode and " worm bag " (worm bag).Adopt stereomicroscope with imaging software beautiful nematode is carried out the video recording of 3min, carry out the mensuration of following index:
(1) steroid hormone is to the influence of beautiful nematode genital system:
A, from the beautiful nematode larval first phase (L1 phase) needed time when beginning to lay eggs.
B, offspring's number: i.e. all ovum numbers in whole egg-laying period.
The time interval that C, generation time: P0 is laid eggs and laid eggs for adult to its F1 for adult.
(2) steroid hormone is to the influence of beautiful nematode locomitivity:
A, head hunting frequency: will grow for the nematode of adult and choose in the solution, and, write down the number of times (swinging as 1 head) of nematode head swing in 1min when the health bending reaches long half of body through the recovery of 1min.
B, health corner frequency: will grow for the nematode of adult and choose on the NGM medium that does not have an OP50, and note the crooked number of times of health in 20 s.Suppose that along the direction of antlia be the y axle, health is defined as 1 health bending along 1 change on the corresponding x direction of principal axis in the nematode crawling process so.
(3) steroid hormone influence that beautiful nematode is grown:
A, life-span: with initial 0 day of nematode life of line eggs hatching beginning to be designated as, to stop with getting the terminal light reactionless nematode life that is designated as of polypide of visiting of worm device.The nematode lifetime is designated as nematode from the initial time that stops to life of life.
B, the long mensuration of body: the nematode health is stiff, and the body when not having bending is long.
5, repeat 1,2,3,4 steps, and the open-assembly time of beautiful nematode is revised as 12h, 18h, 24h respectively.Obtain to expose 12h, steroid hormone is to the result that influences of beautiful nematode each item physical signs; Expose 18h, steroid hormone is to the result that influences of beautiful nematode each item physical signs; Expose 24h, steroid hormone is to the result that influences of beautiful nematode each item physical signs.
6, adopt software Origin7.5 that data are carried out significance analysis; Have significant difference if be exposed to the physical signs of the beautiful nematode in the steroid hormone with the physical signs contrast that is not exposed to the beautiful nematode in the steroid hormone, explain that then steroid hormone has safety hazards to beautiful nematode; If there is not significant difference, explain that then steroid hormone does not have safety hazards to beautiful nematode.
Embodiment 4: set up steroid hormone living model in the chicken feed with beautiful nematode
1, obtain worm liquid of the synchronized beautiful nematode larval first phase (L1 phase):
The beautiful nematode hermaphroditism adult in the egg-laying period is just washed the centrifugal 1min of 1500rpm, supernatant discarded from 90mm NGM culture plate with the M9 buffer solution.As above method is settled to 500 μ l in 1.5ml EP pipe with the M9 buffer solution that the M9 buffer solution for cleaning will contain beautiful nematode after once again.Add lysate 500 μ l, the centrifugal 3min of 1500rpm behind the mixing 3-5min that turns upside down, supernatant discarded is collected bottom precipitation.Deposition is used aseptic S-Medium instead and is cleaned 2-3 time repeatedly to pH value to 7.0, and beautiful nematode polypide is by cracking and worm's ovum is still intact at this moment.The aseptic S-Medium solution that will contain beautiful nematode worm's ovum is put in 20 ℃ and cultivates 8-12h.Owing to there is not food, egg hatch can all stop at the larva first phase (L1 phase) later on, and the centrifugal 3min of 1500rpm collects larva.
2, accurately take by weighing 5g (being accurate to 0.1g) feed in 50mL tool plug centrifuge tube, add the 50mL chloroform, shaken is also carried out natural evaporation.Add 1mL 100% alcohol dissolving steroidal compounds (concentrating 1000 times) again.Take out supernatant solution, be the steroid hormone extract.The liquid that is extracted is put as in the fume hood that it is air-dry, and the use concentration expressed in percentage by volume is 1% dmso solution residue, and is concentrated to suitable concentration.
3, the beautiful nematode larval first phase (L1 phase) worm liquid is exposed in the steroid hormone extract:
Beautiful nematode adopts the liquid exposure method to expose, and two groups of samples are set, and first group is exposed group: get 96 orifice plates; Every hole adds the worm liquid 100 μ l that are in the larva first phase (L1 phase) that 1 collection step obtains, and E.coli OP50 is to OD600=0.250 μ l, liquid NGM culture fluid 49 μ l; Steroid hormone extract 1 μ l; Establish altogether four parallel, 20 ℃ are taken out sample with liquid-transfering gun four parallel inserting in the same 15ml centrifuge tube respectively after expose cultivating 6h.Second group is the blank group: get 96 orifice plates, every hole adds the worm liquid 100 μ l that are in the larva first phase (L1 phase) that 1 collection step obtains, and E.coli OP50 is to OD600=0.2 50 μ l, liquid NGM culture fluid 50 μ l.If four parallel, after cultivating 6h, 20 ℃ of exposures with liquid-transfering gun four parallel sample are taken out four parallel inserting in the same 15ml centrifuge tube respectively.
4, in centrifuge tube, add 4ml M9 buffer solution respectively, leave standstill 10min after shaking up, remove supernatant, add 4ml M9 buffer solution again, leave standstill 10min after shaking up, remove supernatant again, add an amount of M9 buffer solution again and shake up.Taking out 100 μ l in each sample coats on the NGM solid culture medium that contains 150 μ l E.coli OP50; 20 ℃ of cultivations are long till the length that can choose up to nematode; Nematode is chosen, is put on the new NGM solid culture medium that contains 150 μ l E.coli OP50, this moment each sample establish again four parallel; Put into ten nematodes during each is parallel; 20 ℃ of cultivations are every other day chosen nematode to new containing on the 150 μ l E.coli OP50 NGM solid culture mediums, finish up to the egg-laying period.The nematode of losing, should from statistical data, get rid of because of climbing to culture dish wall dead nematode and " worm bag " (worm bag).Adopt stereomicroscope with imaging software beautiful nematode is carried out the video recording of 3min, carry out the mensuration of following index:
(1) steroid hormone is to the influence of beautiful nematode genital system:
A, from the beautiful nematode larval first phase (L1 phase) needed time when beginning to lay eggs.
B, offspring's number: i.e. all ovum numbers in whole egg-laying period.
The time interval that C, generation time: P0 is laid eggs and laid eggs for adult to its F1 for adult.
(2) steroid hormone is to the influence of beautiful nematode locomitivity:
A, head hunting frequency: will grow for the nematode of adult and choose in the solution, and, write down the number of times (swinging as 1 head) of nematode head swing in 1min when the health bending reaches long half of body through the recovery of 1min.
B, health corner frequency: will grow for the nematode of adult and choose on the NGM medium that does not have an OP50, and note the crooked number of times of health in the 20s.Suppose that along the direction of antlia be the y axle, health is defined as 1 health bending along 1 change on the corresponding x direction of principal axis in the nematode crawling process so.
(3) steroid hormone influence that beautiful nematode is grown:
A, life-span: with initial 0 day of nematode life of line eggs hatching beginning to be designated as, to stop with getting the terminal light reactionless nematode life that is designated as of polypide of visiting of worm device.The nematode lifetime is designated as nematode from the initial time that stops to life of life.
B, the long mensuration of body: the nematode health is stiff, and the body when not having bending is long.
5, repeat 1,2,3,4 steps, and the open-assembly time of beautiful nematode is revised as 12h, 18h, 24h respectively.Obtain to expose 12h, steroid hormone is to the result that influences of beautiful nematode each item physical signs; Expose 18h, steroid hormone is to the result that influences of beautiful nematode each item physical signs; Expose 24h, steroid hormone is to the result that influences of beautiful nematode each item physical signs.
6, adopt software Origin7.5 that data are carried out significance analysis; Have significant difference if be exposed to the physical signs of the beautiful nematode in the steroid hormone with the physical signs contrast that is not exposed to the beautiful nematode in the steroid hormone, explain that then steroid hormone has safety hazards to beautiful nematode; If there is not significant difference, explain that then steroid hormone does not have safety hazards to beautiful nematode.
Claims (1)
1. method that steroid hormone is detected with beautiful nematode living model, its method is:
A, utilize beautiful nematode living model that the pure article of steroid hormone are carried out the method that safety hazards detects
A, obtain synchronized beautiful nematode larval first phase worm liquid, the beautiful nematode hermaphroditism adult in the egg-laying period is just washed the centrifugal 1min of 1500rpm, supernatant discarded with the M9 buffer solution from the NGM culture plate; As above method will contain beautiful nematode after once with the M9 buffer solution for cleaning again M9buffer solution constant volume in container, and add the equal-volume lysate, the centrifugal 3min of 1500rpm behind the mixing 3-5min that turns upside down, supernatant discarded is collected bottom precipitation; Deposition is used aseptic S-Medium instead and is cleaned 2-3 time repeatedly and reach neutrality to pH value, and at this moment beautiful nematode polypide is by cracking and worm's ovum is still intact; The aseptic S-Medium solution that will contain beautiful nematode worm's ovum is put in 16 ℃-25 ℃ cultivation 8-12h in the biochemical incubator; Owing to there is not food, egg hatch can all stop at the beautiful nematode larval first phase later on, and the centrifugal 3min of 1500rpm collects larva, and is subsequent use;
B, steroid hormone is dissolved in concentration expressed in percentage by volume is in 1% the dimethyl sulfoxide (DMSO);
C, beautiful nematode larval first phase worm liquid is exposed in the pure article of steroid hormone
Beautiful nematode adopts the liquid exposure method to expose; Two groups of samples are set; First group is exposed group, and with aseptic Tissue Culture Plate, every hole adds that a collection step obtains is in the first-phase worm liquid of beautiful nematode larval and E.coli OP50 and liquid NGM culture fluid and the pure article of steroid hormone; And mixing, the final concentration of steroid hormone is 0.1ng/ml-20ng/ml; Second group is the blank group, and with aseptic Tissue Culture Plate, every hole adds that a collection step obtains is in the first-phase worm liquid of beautiful nematode larval and E.coli OP50 and liquid NGM culture fluid; These two groups of samples are put into 16 ℃-25 ℃ of biochemical incubators expose and with liquid-transfering gun sample is taken out respectively after cultivating 6h, place centrifuge tube;
D, in the centrifuge tube of two groups of samples, add the M9 buffer solution respectively, leave standstill 10min after shaking up, remove supernatant, add the M9 buffer solution again, leave standstill 10min after shaking up, remove supernatant again, add the M9 buffer solution again and shake up; Take out in the sample and coat on the NGM solid culture medium that contains E.coli OP50; In 16 ℃-25 ℃ of biochemical incubators, cultivate up to nematode long till the length that can choose; Nematode is chosen, put on the new NGM solid culture medium that contains E.coli OP50, cultivate in 16 ℃-25 ℃ of the biochemical incubators; Every other day nematode is chosen to new containing on the E.coli OP50NGM solid culture medium, finished up to the egg-laying period; The nematode of losing, should from statistical data, get rid of because of climbing to culture dish wall dead nematode and worm bag; Adopt stereomicroscope with imaging software beautiful nematode is recorded a video, carry out the mensuration of following index
(1) steroid hormone is to the influence of beautiful nematode genital system
(A), from beautiful nematode larval first phase larva needed time when beginning to lay eggs;
(B), offspring's number, offspring's number is all ovum numbers in whole egg-laying period;
(C), generation time, the time interval that P0 is laid eggs and laid eggs for adult to its F1 for adult;
(2) steroid hormone is to the influence of beautiful nematode locomitivity
(A), the head hunting frequency, choose in the solution growing for the nematode of adult, through the recovery of 1min, the number of times of record nematode head swing in 1min is swung as 1 head when the health bending reaches long half of body;
(B), health corner frequency; Choose on the NGM medium that does not have an OP50 growing for the nematode of adult; Note the crooked number of times of health in the 20s; Suppose that along the direction of antlia be the y axle, health is defined as 1 health bending along 1 change on the corresponding x direction of principal axis in the nematode crawling process so;
(3) steroid hormone influence that beautiful nematode is grown
(A), the life-span, begin to be designated as initial 0 day of nematode life with line eggs hatching, so that the worm device is terminal gently visits that polypide is reactionless to be designated as the termination of nematode life with getting; The nematode lifetime is designated as nematode from the initial time that stops to life of life;
(B), the long mensuration of body, the nematode health is stiff, the body that does not have when crooked is long;
E, repeat a, b, c, d step, and the open-assembly time of beautiful nematode is revised as 12h, 18h, 24h respectively; Obtain to expose 12h, steroid hormone is to the result that influences of beautiful nematode each item physical signs; Expose 18h, steroid hormone is to the result that influences of beautiful nematode each item physical signs; Expose 24h, steroid hormone is to the result that influences of beautiful nematode each item physical signs;
F, employing software Origin7.5 carry out significance analysis to data, have significant difference if expose the physical signs of the beautiful nematode in back with the preceding physical signs of exposure, explain that then steroid hormone has safety hazards to beautiful nematode; If there is not significant difference, explain that then steroid hormone does not have safety hazards to beautiful nematode;
B, utilize beautiful nematode living model that steroid hormone pollutant in the food is carried out the method that safety hazards detects
The worm liquid of the beautiful nematode larval first phase of synchronization that step a in a, the repeat techniques option A obtains;
B, fresh experiment is cleaned up with food,,, place homogenizer to be twisted into the mud shape with filter paper filter solid carbon dioxide branch with the scalpel section; Place centrifuge tube, add concentration expressed in percentage by volume and be in 1% the dimethyl sulfoxide (DMSO), stir with glass rod; Vortex oscillation lmin places 55 ℃ of water-baths to be incubated 20min, takes out vortex oscillation 20min; With the centrifugal 20min of 3000rpm, the sucking-off supernatant is the steroid hormone extract, and to be concentrated to concentration be more than 80%;
C, beautiful nematode larval first phase worm liquid is exposed in the steroid hormone extract
Beautiful nematode adopts the liquid exposure method to expose; Two groups of samples are set, and first group is exposed group, with aseptic Tissue Culture Plate; Every hole add obtain be in the first-phase worm liquid of beautiful nematode larval and E.coli OP50 and liquid NGM culture fluid and steroid hormone extract and mixing; Second group is the blank group; With aseptic Tissue Culture Plate; Every hole add obtain be in the first-phase worm liquid of beautiful nematode larval and E.coli OP50 and liquid NGM culture fluid; These two groups of samples are put into 16 ℃-25 ℃ of biochemical incubators expose and with liquid-transfering gun sample is taken out respectively after cultivating 6h, place centrifuge tube;
Steps d, e, f in d, the repeat techniques option A.
C, utilize beautiful nematode living model that steroid hormone pollutant in the feed is carried out the method that safety hazards detects
Step a in a, the repeat techniques option A obtains synchronized beautiful nematode larval first phase worm liquid;
B, get feed in tool plug centrifuge tube, add chloroform, shaken is also carried out natural evaporation, adds 100% alcohol dissolving steroidal compounds again and concentrates 1000 times, takes out supernatant solution, is the steroid hormone extract; The liquid that is extracted is put as in the fume hood that it is air-dry, and using concentration expressed in percentage by volume is 1% dmso solution residue, and to be concentrated to concentration be more than 80%;
C, beautiful nematode larval first phase worm liquid is exposed in the steroid hormone extract
Beautiful nematode adopts the liquid exposure method to expose; Two groups of samples are set, and first group is exposed group, with aseptic Tissue Culture Plate; Every hole add obtain be in the first-phase worm liquid of beautiful nematode larval and E.coliOP50 and liquid NGM culture fluid and steroid hormone extract and mixing; Second group is the blank group; With aseptic Tissue Culture Plate; Every hole add obtain be in the first-phase worm liquid of beautiful nematode larval and E.coli OP50 and liquid NGM culture fluid; These two groups of samples are put into 16 ℃-25 ℃ of biochemical incubators expose and with liquid-transfering gun sample is taken out respectively after cultivating 6h, place centrifuge tube;
Steps d, e, f in d, the repeat techniques option A.
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