CN102183627A - Method for detecting persistent toxicity of soluble heavy metals by using caenorhabditis elegans - Google Patents
Method for detecting persistent toxicity of soluble heavy metals by using caenorhabditis elegans Download PDFInfo
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Abstract
The invention discloses a method for detecting persistent toxicity of soluble heavy metals by using caenorhabditis elegans, relating to a method of detecting the toxic effect of the soluble heavy metals under the conditions of long term and low concentration by carrying out the persistent exposure covering multiple successive generations of soluble heavy metals through adopting caenorhabditis elegans. In the invention, the egg initiation concentration which does not affect the normal egg-hatch and is in the presence without heavy metals, and the food initiation concentration which can meet the needs of growth and development of eggs are determined; toxicity transmission strength and transmission paths and the like of the heavy metals under the long-term actual envrionemnt exposure are obtained through mutual comparison of same indexes among different generations under the condition that the exposure time, and the exposure doses, particularly the coexistence of foods and heavy metals and other aspects stay closer to the actual environmental exposure condition. The invention makes up the deficiency of the traditional method for testing toxicity by using caenorhabditis elegans, has a clear pertinence, and supplements and extends the toxicity research method which is under the condition of simulating actual environmental exposure.
Description
Technical field
Adopt beautiful nematode to detect the method for soluble heavy metal persistence toxicity, relate to and a kind ofly adopt the persistence that beautiful nematode is contained a plurality of successive generations to soluble heavy metal to expose, thereby detect the method for the poisonous effect of this heavy metal under long-term, low consistency conditions.Belong to technical fields such as environmental protection, ecological toxicology research, ecological risk assessment.
Background technology
Beautiful nematode (Caenorhabditis elegans) variation of environment to external world is very responsive, and environment-stress can change development characteristics and other behaviouristics features such as their reproduction speed, life cycle.Toxicologic study often adopts beautiful nematode as the toxotest model animal, but does not have precedent for the research of low concentration, the persistence toxicity of continuity ground a plurality of generation cycles of exposure.
The method of measuring medicine and personal nursing category material generation toxicity with beautiful nematode possesses some special knowledge, its method relates to the beautiful nematode of different generations through the comparison between the effect after the identical exposure, though can obtain certain comparative result to the poisonous effect of heavy metal between alive generation, but this effect and comparative result thereof still there are differences with respect to true environment and deficiency, mainly show: one, exposure duration discontinuous, each its life cycle of nematode from generation to generation has only a certain period to accept exposure, be not the exposure of full life stage, environmental change is responsive more to external world especially not contain beautiful nematode, stage of development from the worm's ovum to the adult; They are two years old, the full-scale condition that can not reflect food and heavy metal coexistence under the actual environment, there are some researches show that food can change the response of beautiful nematode to heavy metal, do not consider that food can not carry out strong explanation for the risk under the true environment condition to the influence of beautiful nematode.
Summary of the invention
The objective of the invention is to disclose a kind of method that adopts beautiful nematode to detect soluble heavy metal persistence toxicity, specifically be as initial biological subject with worm's ovum, determined not influence the worm's ovum initial concentration of the normal hatching of worm's ovum, and can satisfy the food initial concentration that it grows required, in exposure duration, reconditioning is aspect such as food and heavy metal coexistence closing to reality environmental exposure situation more especially, adopt beautiful nematode to carry out the toxotest method relatively with existing, have clear and definite specific aim, the simulation actual environment is exposed toxicity research method under the situation replenish and expand.
In order to achieve the above object, the present invention adopts the persistence that beautiful nematode is contained a plurality of successive generations to heavy metal to expose, thereby detects the poisonous effect of this heavy metal under long-term, low consistency conditions.The present invention has determined not influence the worm's ovum initial concentration of egg hatch, and can satisfy the food initial concentration that it grows required, the closing to reality environmental exposure situation more at aspects such as exposure duration, reconditioning especially food and heavy metal coexistences.Used beautiful nematode of the present invention and E.coli OP50 are so kind as to give by Developmental Biology research institute of Fudan University.Specifically comprise the steps:
A according to traditional acute toxic test method, as biological subject, records the acute LC of this soluble heavy metal with beautiful nematode adult
50Value is at acute LC
501/10 time of value 4 concentration gradients are set, comprising 3 heavy metal concentrations, 1 blank; Wherein, measure LC
50Concrete steps be: obtain synchronized ovum liquid according to the B step, cultivated on the NGM solid medium of 24h being covered with E.coli OP50 and 37 ℃, place 20 ℃ to cultivate 48h, obtain synchronized beautiful nematode adult, be toxic environment with 96 orifice plates then, in every hole, add 15 beautiful nematode adults, adopt stereomicroscope that the beautiful nematode in 96 orifice plates is observed behind the contamination 24h, polypide is stiff and then assert its death to slight touching is reactionless, write down sum and the death toll of beautiful nematode adult respectively, calculate mortality ratio; If the concentration gradient that sets does not contain 30% to 70% mortality ratio simultaneously, then according to circumstances adjust the concentration gradient of heavy metal: if the mortality ratio that concentration gradient produced that sets is all less than 70%, then keep blank constant, least concentration in the heavy metal concentration gradient is constant, increase the logarithmic interval that waits that is adopted, set higher concentration gradient; If the mortality ratio that concentration gradient produced that sets then keeps blank constant all greater than 30%, and the maximum concentration in the heavy metal concentration gradient is constant, increase the logarithmic interval that waits that is adopted, set lower concentration gradient.And then be toxic environment with 96 orifice plates, in every hole, add 15 beautiful nematode adults, adopt stereomicroscope that mortality ratio is observed behind the contamination 24h; Repeating this operation and contain 30% to 70% simultaneously until the mortality ratio that obtains, is that horizontal ordinate, mortality ratio are ordinate with the concentration logarithm then, adopts method of linear interpolation to obtain its acute LC
50Value;
B, according to classic method, under 20 ℃ of conditions with beautiful culture of nematodes after three days, with the 2mL sterilized water the beautiful nematode of NGM media surface is washed to the centrifuge tube of 15mL tool scale, leave standstill 30min, remove supernatant to worm liquid less than 1.5mL, according to the conventional synchronization method, according to worm liquid: the volume ratio of clorox solution=1: 7, clorox solution is added in the worm liquid, mixing shakes up, reaction 20min, shook up once in per five minutes, kill parent, stay the worm's ovum that clorox solution is had resistivity, the centrifugal 3min of 2500rpm, abandon supernatant, add sterilized water again to centrifugal preceding identical scale, shake up, the centrifugal 3min of 2500rpm abandons supernatant, repeat twice of this operation, in centrifuge tube, add mixing behind the aseptic K-solution that 10mL obtains according to conventional formulation then, the worm's ovum quantity that adopts stereoscope to observe 40 these mixed liquors of μ L, nymph ovum quantity is more than 500 pieces, then in centrifuge tube, add the aseptic K-solution of 2mL mixing more again, observe, nymph ovum quantity is less than 300 pieces, then staticly settles 10min, removes 2mL supernatant mixing again, observe, until being till per 40 μ L contain the 400 pieces of worm's ovums of having an appointment with the worm's ovum concentration dilution, this density can not influence the normal hatching of worm's ovum, and this is first worm's ovum of contaminating, and is labeled as ovum liquid P
0, stand-by;
C, the LB fluid nutrient medium that employing obtains according to conventional formulation is at 37 ℃, under the 200rpm condition E.coliOP50 is cultivated 24h to 48h, pour into behind the mixing in the sterilized 15mL centrifuge tube, the centrifugal 5min of 4000rpm, abandon supernatant, keep the thalline that is sunken to the bottom, add the aseptic K-solution of 8mL mixing, with 24 orifice plates is container, with the enzyme-linked immunosorbent assay instrument is detecting instrument, measure the absorbance of per 300 these mixed liquors of μ L, if absorbance is then centrifugal again with mixed liquor less than 1.1 or greater than 1.3 at the 570nm place, abandon supernatant, keep the thalline that is sunken to the bottom, add 6mL or the aseptic K-solution of 10mL mixing, measure the absorbance of mixed liquor once more at the 570nm place, by the volume that reduces or increase aseptic-K solution absorbance is adjusted between 1.1 and 1.3, this moment, the total amount of E.coli OP50 was about 10
8To 10
9Individual, the bacterium number under this quantity term can be grown for the normal growth of the beautiful nematode worm's ovum of B step gained provides enough food, is labeled as bacterium liquid, stand-by;
D, adopt aseptic 24 transparent orifice plates to contaminate, the bacterium liquid that the C step is obtained shakes up earlier, in every hole of 24 orifice plates, add this bacterium liquid of 300 μ L, the B step is diluted good ovum liquid shake up, in every hole of 24 orifice plates, add this ovum liquid of 200 μ L, 24 orifice plates are divided into 4 row, 6 row, these contain 24 holes of worm's ovum, and every row is contaminated to beautiful nematode as one group, 4 row 4 groups of 4 concentration gradients determining corresponding to the A step totally; Adding the aseptic K-solution of 1mL between the Kong Yukong of 24 orifice plates with during reducing contamination, evaporation is to the influence of contamination system; Can be according to actual needs the number and the relevant layout of 24 orifice plates that adopted be adjusted; 24 orifice plates are placed under 20 ℃ of conditions cultivate and pick up counting;
E, behind the 96h of D step timing, the beautiful nematode that pipettes at random from every row in 1 hole is used to measure index of correlation, all is labeled as P
0Each desired value;
F, the mixed liquor of 4 concentration gradient correspondences in 24 orifice plates is drawn to respectively in 4 aseptic 15mL centrifuge tubes, and wash residual polypide in each hole of 24 orifice plates with the aseptic K-solution of 1mL, and washing fluid is transferred in the corresponding centrifuge tube, staticly settle 15min, abandon supernatant and keep precipitation, according to the conventional synchronization method, add clorox solution to the 12mL scale mark, mixing shakes up, and reaction 20min shook up once in per five minutes, kill parent, stay the worm's ovum that clorox solution is had resistivity, the centrifugal 3min of 2500rpm abandons supernatant, add sterilized water again to centrifugal preceding identical scale, shake up, the centrifugal 3min of 2500rpm abandons supernatant, repeat twice of this operation, according to the method among the step B, be dilution then, the worm's ovum concentration in 4 centrifuge tubes diluted respectively for per 40 μ L contain 400 pieces of worm's ovums with aseptic K-solution, this batch worm's ovum is the first filial generation worm's ovum in female generation, is labeled as ovum liquid F
1, stand-by; Repeating step C obtains bacterium liquid, according to identical with steps A concentration gradient, repeating step D, the E of being tried, obtains to be labeled as F
1Each desired value;
G, repeating step F obtains second filial F
2, F3 F
3Until x for filial generation F
xEach index;
H, according to the significant difference analysis result of each generation between the desired value of beautiful nematode that obtains in E, F, the G step, be divided into reference to the method for the generation toxicity of measuring medicine and personal nursing category material with beautiful nematode persistence toxicity classification with this soluble heavy metal: do not have clear and definite persistence toxicity, have retardance persistence toxicity, have the toxicity that can be repaired, have clearly and the persistence toxicity that can not repair.
Effect of the present invention and advantage are:
1. measuring medicine with the beautiful nematode of previous employing compares with the method for personal care articles generation toxicity, step of the present invention still less, the operation simpler, not only kept exposure level near actual environment concentration, and realized the continuity of exposure duration, make the whole life of beautiful nematode in a plurality of successive generations all be encompassed in exposure duration, especially contained beautiful nematode to external world environmental change responsive more, the stage of development from the worm's ovum to the adult, exposure duration of the present invention is more near the exposure under the actual environment condition.
2. measuring medicine with the beautiful nematode of previous employing compares with the method for personal care articles generation toxicity, the present invention considers that food can change the research of beautiful nematode counterweight metallic response, determined not influence the worm's ovum initial concentration of hatching, and can satisfy the food initial concentration that it grows required, realized the coexistence of food and heavy metal, exposed situation more near the exposure under the actual environment condition.
3. this method has been acted on the availability of beautiful nematode behaviouristics, auxology, neurology etc. being enriched index, and beautiful nematode individual level, Physiology and biochemistry level, the multifaceted every toxicity index of molecular water equality all can be compatible mutually with this method.Beautiful nematode through near actual environment have food coexistence, low concentration, for a long time expose after, different generations is (female for P
0, first filial generation F
1, second filial F
2, F3 F
3Until x for filial generation F
x) between the mutual comparative result of identical index, whether the toxicity that not only can obtain this heavy metal passes to the offspring, the power that this kind toxicity is transmitted, the approach of transmission, and different generations is to the susceptibility of this heavy metal result such as whether change, the feasible toxicity research that is carried out is adding system, comprehensive more, provides clear and definite direction for order and the mechanism of judging toxicity generation effect; And can provide more rationally, expose near actual environment more the data basis of situation, and then provide data more reliably for the ecological risk of estimating this heavy metal species for the evaluation of this heavy metal and management.
Embodiment
Embodiment 1
Preparation LB (Luria Bertani) nutrient culture media, NGM (Nematode Growth Medium) solid medium, K-solution and clorox solution.
1. according to " molecular cloning test guide " (J. Sa nurse Brooker, D.W. Russell etc.) preparation LB nutrient culture media (1L): 10g tryptone, 5g yeast extract, 10g sodium chloride, with 1mol/L NaOH solution the pH value is adjusted to 7.0, at 121 ℃, the 0.105MPa 20min that sterilizes.In addition: preparation 1mol/L NaOH solution (100mL): 4g NaOH, 100mL water.
2. the paper of delivering on the Genetics publication in 1974 according to S.Brenner (The genetics ofCaenorhabditis elegans) is prepared NGM solid medium (1L): the K of 17g agar powder, 2.5g peptone, 3g sodium chloride, 25mL pH=6.0
2HPO
4-KH
2PO
4Solution; 121 ℃, 0.105MPa autoclaving 20min when being cooled to 50 ℃ of left and right sides, add the 1mol/L MgSO that crosses through the suction filtration sterilization treatment
4, 1mol/LCaCl
2, each 1mL of 5mg/mL cholesterol/ethanolic solution, pour into after mixing in the double dish after the sterilization, leave standstill and be cooled to after room temperature solidifies, wait to inoculate.In addition: preparation 1mol/L K
2HPO
4-KH
2PO
4Damping fluid (pH=6.0,150mL): 11.4g K
2HPO
43H
2O, 50mL distilled water; 6.8g KH
2PO
4, 50mL distilled water; Is the damping fluid that 2: 1 ratios are made into pH=6.0 in the former than the latter.Preparation cholesterol solution (100mL): 0.5g cholesterol, 100mL anhydrous alcohol solution; The suction filtration sterilization.Preparation 1mol/L MgSO
4(100mL): 24.6g MgSO
47H
2O, 100mL distilled water; The suction filtration sterilization.Preparation 1mol/L CaCl
2: 11.1g CaCl
2, 100mL distilled water; The suction filtration sterilization.Suction filtration degerming: with the cellulose nitrate membrane filtration mushroom of aseptic Φ 13mm aperture 0.45 μ m.With filter membrane be contained in capacity that high-temperature sterilization handled be the glass syringe of 5mL by the needle tubing place, with the liquid to be filtered syringe of packing into, push injection device piston rod, the solution that extrudes liquid film are the solution after the suction filtration degerming.
3. the paper of on nineteen ninety Environemtnal Toxicology and Chemistry publication, delivering according to people such as P.L.Williams (Aquatic toxicity testing using the nematode, Caenorhabditiselegans) preparation K-solution (1L): 3.00g NaCl, 2.36g KCl; At 121 ℃, the 0.105MPa 20min that sterilizes.
4. the paper of delivering on Proceedings of the National Academyof Sciences publication in 1979 according to people such as S.W.Emmons (An analysis of the constancy of DNA sequencesduring development and evolution of the nematode Caeno
Return in the LB nutrient culture media of room temperature after Escherichia coli OP50 (E.coli OP50) is seeded to sterilization, cultivate 24h for 37 ℃ and can use.Cultured OP50 E.coli is seeded on the NGM nutrient culture media, cultivates 24h for 37 ℃ and can use; Beautiful nematode is seeded on this NGM nutrient culture media 20 ℃ of conventional cultivations.Used beautiful nematode of the present invention and E.coli OP50 are so kind as to give by Developmental Biology research institute of Fudan University.
Adopt beautiful nematode to detect the method for soluble heavy metal persistence toxicity, concrete steps are as follows:
A according to traditional acute toxic test method, as biological subject, records the acute LC of this heavy metal with beautiful nematode adult
50Value is at acute LC
501/10 time of value 8 concentration gradients are set, comprising 7 heavy metal concentrations, 1 blank; Wherein, measure LC
50Concrete steps be: obtain synchronized ovum liquid according to the B step, on the NGM solid medium that is covered with E.coli OP50 and 37 ℃ of cultivation 24h, cultivate 48h for 20 ℃, obtain synchronized beautiful nematode adult, be toxic environment with 96 orifice plates then, in every hole, add 15 beautiful nematode adults, adopt stereomicroscope that the beautiful nematode in 96 orifice plates is observed behind the contamination 24h, polypide is stiff and then assert its death to slight touching is reactionless, write down sum and the death toll of beautiful nematode adult respectively, calculate mortality ratio; If the concentration gradient that sets does not contain 30% to 70% mortality ratio simultaneously, then according to circumstances adjust the concentration gradient of heavy metal: if the mortality ratio that concentration gradient produced that sets is all less than 70%, then keep blank constant, least concentration in the heavy metal concentration gradient is constant, increase the logarithmic interval that waits that is adopted, set higher concentration gradient; If the mortality ratio that concentration gradient produced that sets then keeps blank constant all greater than 30%, and the maximum concentration in the heavy metal concentration gradient is constant, increase the logarithmic interval that waits that is adopted, set lower concentration gradient.And then be toxic environment with 96 orifice plates, in every hole, add 15 beautiful nematode adults, adopt stereomicroscope that mortality ratio is observed behind the contamination 24h; Repeating this operation and contain 30% to 70% simultaneously until the mortality ratio that obtains, is that horizontal ordinate, mortality ratio are ordinate with the concentration logarithm then, adopts method of linear interpolation to obtain its acute LC
50Value;
B, according to classic method, under 20 ℃ of conditions, with beautiful nematode is conventional cultivate three days after, with the 2mL sterilized water the beautiful nematode of NGM media surface is washed to the centrifuge tube of 15mL tool scale, leave standstill 30min, remove supernatant to worm liquid less than 1.5mL, according to the conventional synchronization method, according to worm liquid: the volume ratio of clorox solution=1: 7, clorox solution is added in the worm liquid, mixing shakes up, reaction 20min, shook up once in per five minutes, kill parent, stay the worm's ovum that clorox solution is had resistivity, the centrifugal 3min of 2500rpm, abandon supernatant, add sterilized water again, shake up to centrifugal preceding identical scale, the centrifugal 3min of 2500rpm, abandon supernatant, repeat this operation twice, in centrifuge tube, add the aseptic K-solution of 10mL mixing then, the worm's ovum quantity that adopts stereoscope to observe 40 these mixed liquors of μ L, nymph ovum quantity then adds the aseptic K-solution of 2mL mixing more again more than 500 pieces in centrifuge tube, observe, nymph ovum quantity is less than 300 pieces, then staticly settle 10min, remove 2mL supernatant mixing again, observe, until being that this density can not influence the normal hatching of worm's ovum till per 40 μ L contained the 400 pieces of worm's ovums of having an appointment with the worm's ovum concentration dilution, this batch worm's ovum is first worm's ovum of contaminating, and is labeled as ovum liquid P
0, stand-by;
C, the LB fluid nutrient medium that employing obtains according to conventional formulation is at 37 ℃, under the 200rpm condition E.coliOP50 is cultivated 24h to 48h, pour into behind the mixing in the sterilized 15mL centrifuge tube, the centrifugal 5min of 4000rpm, abandon supernatant, keep the thalline that is sunken to the bottom, add the aseptic K-solution of 8mL mixing, with 24 orifice plates is container, with the enzyme-linked immunosorbent assay instrument is detecting instrument, measure the absorbance of per 300 these mixed liquors of μ L, if absorbance is then centrifugal again with mixed liquor less than 1.1 or greater than 1.3 at the 570nm place, abandon supernatant, keep the thalline that is sunken to the bottom, add 6mL or the aseptic K-solution of 10mL mixing, measure the absorbance of mixed liquor once more at the 570nm place, by the volume that reduces or increase aseptic-K solution absorbance is adjusted between 1.1 and 1.3, this moment, the total amount of E.coli OP50 was about 10
8To 10
9Individual, the bacterium number under this quantity term can be grown for the normal growth of the beautiful nematode worm's ovum of B step gained provides enough food, is labeled as bacterium liquid, stand-by;
D, 24 orifice plates are divided into 4 row, 6 row, adopt two aseptic 24 transparent orifice plates to contaminate, the bacterium liquid that the C step is obtained shakes up earlier, add this bacterium liquid of 300 μ L in totally 48 holes to two 24 orifice plates, the B step is diluted good ovum liquid to be shaken up, totally 12 holes, two plates add this ovum liquid of 200 μ L in totally 24 holes to the first three columns with each 24 orifice plate, these contain 24 holes of worm's ovum,, as one group, two plates totally 8 groups of 8 concentration gradients determining corresponding to the A step beautiful nematode is contaminated with 3 holes of delegation; Being listed as to back three of each 24 orifice plate, totally 12 holes, two plates add the aseptic K-solution of 200 μ L liquid as a supplement in totally 24 holes, 24 holes of these no worm's ovums, as one group, two plates totally 8 groups of 8 concentration gradients determining corresponding to the A step, be used to provide bacterium liquid to be subjected to the contrast that changes under the strip spare with 3 holes of delegation at this; Then with the ready heavy metal of C step with concentration gradient of every row, two plates totally 8 concentration gradients, add 500 μ L heavy metals respectively in totally 48 holes to two plates, utilize microplate reader to measure the absorbance of every hole behind the mixing immediately at the 570nm place, be designated as OD
BeginningAdding the aseptic K-solution of 1mL between the Kong Yukong of 24 orifice plates with during reducing contamination, evaporation is to the influence of contamination system; 24 orifice plates are placed under 20 ℃ of conditions cultivate and pick up counting;
E behind the 96h of D step timing, utilizes microplate reader to measure the absorbance of every hole at the 570nm place, is designated as OD
The end, the changing value of each hole OD value is Δ OD=OD
Beginning-OD
The end, contain 8 gradients of ovum liquid in the D step, the Δ OD in totally 24 holes all will deduct same concentration gradient, not contain the Δ OD of the reference opening of ovum liquid, is designated as dietary amount inhibiting rate (P
0); From each concentration gradient, contain the beautiful nematode that pipettes at random in three holes of ovum liquid in 1 hole, measure the body size of beautiful nematode, the health corner frequency, the detection of indexs such as reverse movement, concrete implementation step is: take a morsel beautiful nematode respectively with liquid-transfering gun to the 1.5mL centrifuge tube from 8 groups of concentration, precipitation 15min, adopt liquid-transfering gun to remove supernatant, add sterile distilled water again and clean OP50E.coli and the heavy metal that beautiful nematode is sticked on one's body, precipitation 15min, the beautiful nematode in bottom is transferred on the NGM nutrient culture media of no OP50E.coli, place 20 ℃ of incubators, behind the 2h, after water evaporates and beautiful nematode have conformed, the imaging software that adopts stereomicroscope to attach is taken a picture to beautiful nematode and carried out the video recording of 60sec, and is long to the body of beautiful nematode according to document, the health corner frequency, indexs such as reverse movement frequency detect.Every index is all represented with the ratio of the identical index of beautiful nematode of the white control group of its duty.Every index is labeled as the " long (P of body respectively
0) ", " health corner frequency (P
0) ", " reverse movement frequency (P
0) ";
F, to be drawn to respectively through the mixed liquor that contains 8 concentration gradient correspondences of ovum liquid in 2 24 orifice plates after the E step in 8 aseptic 15mL centrifuge tubes, and wash residual polypide in each hole of 24 orifice plates with the aseptic K-solution of 1mL, and washing fluid is transferred in the corresponding centrifuge tube, staticly settle 15min, abandon supernatant and keep precipitation, add clorox solution to the 12mL scale mark, mixing shakes up, reaction 20min, shook up once in per five minutes, and killed parent, stay the worm's ovum that clorox solution is had resistivity, the centrifugal 3min of 2500rpm, abandon supernatant, add sterilized water again, shake up to centrifugal preceding identical scale, the centrifugal 3min of 2500rpm, abandon supernatant, repeat this operation twice, then according to the method among the step B, with aseptic K-solution is dilution, worm's ovum concentration in 8 centrifuge tubes is diluted respectively for per 40 μ L contain 400 pieces of worm's ovums, and this batch worm's ovum is the first filial generation worm's ovum in female generation, is labeled as ovum liquid F
1, stand-by; Repeating step C obtains bacterium liquid, according to identical with steps A concentration gradient, repeating step D, the E of being tried, obtains to be labeled as F
1Each desired value, comprise " dietary amount inhibiting rate (F
1) ", " long (F of body
1) ", " health corner frequency (F
1) ", " reverse movement frequency (F
1) ";
G, repeating step F obtains second filial F
2Each desired value, comprise " dietary amount inhibiting rate (F
2) ", " long (F of body
2) ", " health corner frequency (F
2) ", " reverse movement frequency (F
2) "; F3 F
3Every desired value, comprise " dietary amount inhibiting rate (F
3) ", " long (F of body
3) ", " health corner frequency (F
3) ", " reverse movement frequency (F
3) "; And sub four generation F
4Each index, comprise " dietary amount inhibiting rate (F
4) ", " long (F of body
4) ", " health corner frequency (F
4) ", " reverse movement frequency (F
4) " etc.;
H, according to the method for measuring the generation toxicity of medicine and personal nursing category material with beautiful nematode the persistence toxicity of heavy metal is estimated, data analysis that employing U.S. OriginLab company releases and graphics software Origin 7.5 and the computer that satisfies this software requirement are to comprising first filial generation F
1, second filial F
2, F3 F
3Until x for filial generation F
xDeng toxicity result and beautiful nematode mother for P
0Toxicity result compare and carry out significance analysis, concrete implementation step is as follows: " dietary amount inhibiting rate (the P that E, F, G step are obtained
0) ", " dietary amount inhibiting rate (F
1) ", " dietary amount inhibiting rate (F
2) ", " dietary amount inhibiting rate (F
3) " data inputs Origin 7.5 data lists, under statistics, open the one-way anova in the drop-down button of anova, from candidate list with " dietary amount inhibiting rate (P
0) " and " dietary amount inhibiting rate (F
1) " data select, and " conspicuousness parameter " is set at 0.05, click " calculating " can obtain dietary amount inhibiting rate (F
1) and dietary amount inhibiting rate (P
0) significant difference result; If result of calculation is shown as " At the 0.05level, thepopulation means are not significantly different. ", i.e. " there is not significant difference in the two under 0.05 level "; If the result is shown as " At the 0.05level, the population means aresignificantly different. ", i.e. " there is significant difference in the two under 0.05 level ".And then under statistics, open one-way anova in the drop-down button of anova, from candidate list with " dietary amount inhibiting rate (P
0) " and " dietary amount inhibiting rate (F
2) " data select, and " conspicuousness parameter " is set at 0.05, click " calculating " can obtain dietary amount inhibiting rate (F
2) and dietary amount inhibiting rate (P
0) significant difference result; By that analogy, obtain dietary amount inhibiting rate (F
3) and dietary amount inhibiting rate (P
0) significant difference result and dietary amount inhibiting rate (F
3) and dietary amount inhibiting rate (P
0) significant difference result.If the comparative result of " dietary amount inhibiting rate " comprises " F
1With P
0", " F
2With P
0", " F
3With P
0", " F
4With P
0", all do not obtain the result of significant difference, illustrate that then this heavy metal does not have clear and definite persistence toxicity to this index; If " F
1With P
0", " F
2With P
0" comparative result be " not having significant difference ", and " F
3With P
0", " F
4With P
0" comparative result be " having significant difference ", and F
3, F
4Show even more serious poisonous substance depression effect, illustrate that then this heavy metal has the persistence toxicity of retardance to this index; If " F
1With P
0", " F
2With P
0" comparative result be " having significant difference ", and " F
3With P
0", " F
4With P
0" comparative result be " not having significant difference ", and F
3, F
4Show comparatively slight poisonous substance depression effect, illustrate that then this heavy metal has the toxicity that can be repaired or adapt to by beautiful nematode to this index; If " F
1With P
0", " F
2With P
0", " F
3With P
0", " F
4With P
0" comparative result be " having significant difference ", and " F
2With F
1", " F
3With F
1", " F
4With F
1" comparative result, " F
3With F
2", " F
4With F
2" comparative result, " F
4With F
3" comparative result also all be " having significant difference ", and P
0, F
1, F
2, F
3, F
4The poisonous substance depression effect that is subjected to is also more serious, illustrates that then this heavy metal has persistence toxicity clear and definite, that can not repair to this index.
Other index is carried out identical significant difference analysis, and persistence toxicity is estimated.Take all factors into consideration the evaluation result of every index to persistence toxicity, after judging that the persistence that is subjected to this heavy metal exposes, the sequencing of the poisonous effect that beautiful nematode showed, diet at first is affected, behaviouristics is active and then be affected, perhaps the behaviouristics activity at first is affected, diet is affected then, perhaps other the order, sequencing according to different poisonous effects, can infer that this heavy metal is the poisonous effect that produces by diet, body surface or other approach, the mechanism of toxication provider who produces this persistence toxicity for research to.
Claims (1)
1. adopt beautiful nematode to detect the method for soluble heavy metal persistence toxicity, comprise A,, as biological subject, record the acute LC of this heavy metal with beautiful nematode adult according to the acute toxic test method
50Value is at acute LC
503 heavy metal concentrations and 1 blank are set, it is characterized in that for 1/10 time of value:
B, utilize the 15mL centrifuge tube of aseptic tool scale, adopt method for synchronizing to obtain beautiful nematode worm's ovum, in the 15mL centrifuge tube, add the aseptic K-solution of 10mL then, mixing, the worm's ovum quantity that adopts stereoscope to observe 40 these mixed liquors of μ L, nymph ovum quantity is more than 500 pieces, then adds mixing, observation again behind the aseptic K-solution of 2mL in centrifuge tube again; Nymph ovum quantity is less than 300 pieces, then staticly settles 10min, mixing, observation again after the removal 2mL supernatant; Until being that this batch worm's ovum was first worm's ovum of contaminating, and is labeled as ovum liquid P till per 40 μ L contained 400 pieces of worm's ovums with the worm's ovum concentration dilution
0, stand-by;
C, adopt the LB fluid nutrient medium at 37 ℃, under the 200rpm condition E.coli OP50 is cultivated 24h to 48h, pour into behind the mixing in the sterilized 15mL centrifuge tube, the centrifugal 5min of 4000rpm, abandon supernatant, keep the thalline that is sunken to the bottom, add the aseptic K-solution of 8mL mixing, with 24 orifice plates is container, with the enzyme-linked immunosorbent assay instrument is detecting instrument, measure the absorbance of per 300 these mixed liquors of μ L, if absorbance is then centrifugal again with mixed liquor less than 1.1 or greater than 1.3 at the 570nm place, abandon supernatant, keep the thalline that is sunken to the bottom, add 6mL or the aseptic K-solution of 10mL mixing, measure the absorbance of mixed liquor once more at the 570nm place, by the volume that reduces or increase aseptic-K solution absorbance is adjusted between 1.1 and 1.3, this moment, the total amount of E.coli OP50 was 10
8To 10
9Individual, the bacterium number under this quantity term can be grown for the normal growth of the beautiful nematode worm's ovum of B step gained provides enough food, is labeled as bacterium liquid, stand-by;
D, adopt aseptic 24 transparent orifice plates to contaminate, the bacterium liquid that the C step is obtained shakes up earlier, in every hole of 24 orifice plates, add this bacterium liquid of 300 μ L, the B step is diluted good ovum liquid shake up, in every hole of 24 orifice plates, add this ovum liquid of 200 μ L, 24 orifice plates are divided into 4 row, 6 row, these contain 24 holes of worm's ovum, and every row is contaminated to beautiful nematode as one group, 4 row 4 groups of 4 concentration gradients determining corresponding to the A step totally; Adding the aseptic K-solution of 1mL between the Kong Yukong of 24 orifice plates with during reducing contamination, evaporation is to the influence of contamination system; Can be according to actual needs the number and the relevant layout of 24 orifice plates that adopted be adjusted; 24 orifice plates are placed under 20 ℃ of conditions cultivate and pick up counting;
E, behind the 96h of D step timing, the beautiful nematode that pipettes at random from every row in 1 hole is used to measure index of correlation, all is labeled as P
0Each desired value;
F, the mixed liquor of 4 concentration gradient correspondences in 24 orifice plates is drawn to respectively in 4 aseptic 15mL centrifuge tubes, wash residual polypide in each hole of 24 orifice plates with the aseptic K-solution of 1mL, and washing fluid is transferred in the corresponding centrifuge tube, staticly settle 15min, abandon supernatant and keep precipitation, adopt method for synchronizing, obtain 4 groups of worm's ovums of 4 concentration gradient correspondences, according to the method among the step B, be dilution then, the worm's ovum concentration in 4 centrifuge tubes diluted respectively for per 40 μ L contain 400 pieces of worm's ovums with aseptic K-solution, this batch worm's ovum is the first filial generation worm's ovum in female generation, is labeled as ovum liquid F
1, stand-by; Repeating step C obtains bacterium liquid, according to identical with steps A concentration gradient, repeating step D, the E of being tried, obtains to be labeled as F
1Each desired value;
G, repeating step F obtains second filial F
2, F3 F
3Until x for filial generation F
xEach index;
H, the significant difference analysis result of each generation between the desired value of beautiful nematode according to obtaining in E, F, the G step is divided into reference to the persistence toxicity classification of classic method with this soluble heavy metal: do not have clear and definite persistence toxicity, have retardance persistence toxicity, have the toxicity that can be repaired, have persistence toxicity clear and definite and that can not repair.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104569330A (en) * | 2014-12-04 | 2015-04-29 | 安徽省农业科学院农业工程研究所 | Caenorhabditis elegans based micro water sample toxicology detection method |
| CN105021772A (en) * | 2015-07-09 | 2015-11-04 | 中国科学院东北地理与农业生态研究所 | Method for detecting copper by pharyngeal motion of Caenorhabditis elegans |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101451983A (en) * | 2008-12-25 | 2009-06-10 | 同济大学 | Method for detecting generation toxicity of medicament and personal cure by Caenorhabditis elegans |
-
2011
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101451983A (en) * | 2008-12-25 | 2009-06-10 | 同济大学 | Method for detecting generation toxicity of medicament and personal cure by Caenorhabditis elegans |
Non-Patent Citations (2)
| Title |
|---|
| 张燕芬: "利用模式动物秀丽线虫建立环境毒物毒性的评估研究体系", 《生态毒理学报》 * |
| 王大勇: "铬暴露导致的秀丽线虫多重毒性的世代间比较", 《生态毒理学报》 * |
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| CN104569330B (en) * | 2014-12-04 | 2017-03-01 | 安徽省农业科学院农业工程研究所 | A kind of micro water sample toxicology detection method based on Caenorhabditis elegans |
| CN105021772A (en) * | 2015-07-09 | 2015-11-04 | 中国科学院东北地理与农业生态研究所 | Method for detecting copper by pharyngeal motion of Caenorhabditis elegans |
| CN106770946A (en) * | 2016-12-14 | 2017-05-31 | 同济大学 | A kind of analysis method of environmental contaminants to C. Elegans Automatic Screening lethal toxicity |
| CN109342709A (en) * | 2018-11-21 | 2019-02-15 | 蚌埠医学院 | A method of based on caenorhabditis elegan detection plasticiser more than generation cumulative toxicity |
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| CN110863030A (en) * | 2019-07-31 | 2020-03-06 | 同济大学 | Method for detecting developmental toxicity by using caenorhabditis elegans |
| CN114085884A (en) * | 2021-11-17 | 2022-02-25 | 中国科学技术大学 | Application of caenorhabditis elegans in detection of nano plastic and nano silver generation combined toxicity |
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