CN104122355B - Method for evaluating kidney toxicity of compounds through detecting contents of creatinine in zebra fish tissues - Google Patents
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Abstract
The invention relates to a method for evaluating kidney toxicity of compounds through detecting the contents of creatinine in zebra fish tissues. The method is applied to kidney toxicity researches. In the researches that utilize a zebra fish embryo model to study the toxic effect of compounds on the kidney, the creatinine component in embryo tissues is extracted, then the creatinine content is measured through a LC-MS (liquid chromatography-mass spectrum) method, the creatinine content can be taken as a detection index for evaluating the embryo kidney functions, and thus a rapid and precise compound kidney toxicity evaluation system with strong specificity is built.
Description
Technical field
The present invention relates to a kind of method by detecting zebra fish tissues creatinine content evaluation compound Toxicity of Kidney, belong to
Toxicology detection field.
Background technology
Kidney is the vitals of body, can discharge interior metabolism product and some wastes, poisonous substance, adjusts body water, electricity
Solution matter balance is it is ensured that the stablizing of organismic internal environment.As the Major excretion organ of body, kidney is highly prone to various metabolism in vivo
Waste, the impact of poisonous substance, these materials often have potential Toxicity of Kidney.With social development, metal, smelting industry, chemical industry
Enterprise increases, in addition medical and various indiscriminate use of pesticide, and environmental pollution increasingly increases, and the chance that crowd contacts nephrotoxicity material increases
Plus, the sickness rate of toxic nephrosis increases substantially.The kidney injury that clinically nephrotoxicant matter leads to, often shows as acute renal
Exhaustion (acute renal failure, arf), accounts for the 5～25% of acute renal failure.Acute renal failure onset is dangerous, mortality rate
Height, needs all the life carry out dialysis treatment or even kidney transplantation patient more, brings huge misery and heavy warp to patient and its family
Ji burden.Therefore, the Toxicity of Kidney effect that fast and effectively appraisement system or evaluation methodology carry out detection compound, centering are established
The early prevention tool significance of toxicity kidney damage.
Research in terms of the existing at present Toxicity of Kidney with regard to compound is many with mammals such as rat, mice, rabbit to be
Model is carried out, and the detection meanss of renal function mainly have blood, urine Biochemical Indexes, and renal histopathology is observed, this
A little animal physique are larger, raise and experimental cost is high, the required cycle is long, the high throughput testing of toxicity of compound difficult to realize.Profit
Carry out the important alternatives that in vitro toxicity analysis is early stage toxicity assessment with cultured cells system, can quickly produce substantial amounts of preliminary
As a result, due to cell in vitro departing from the organismic internal environment of the complexity residing for kidney it is impossible to true reflection each organ is mutual in vivo
The change of the lower renal function of effect, the result being obtained needs the enterprising step card of living Animal Models, therefore, evaluation model
Be selected to realize the bottleneck of toxicity of compound quick detection.
Brachydanio rerio is a kind of tropica minor fresh-water fishes, belongs to Cyprinidae.Its adult fish only about 3-4 centimeter length, egg laying amount is big, and embryo is saturating
Bright and ectogenesises, are highly convenient for operating and observe.Its embryonic development period is short, and many researchs can be carried out in period of embryo.
Brachydanio rerio genome sequence is highly similar to human genome, the multiple important set including cardiovascular system, nervous system
Knit organ in morphosiss, physiological function and pathological reaction similar with the mankind so as to give birth to as a kind of preferable vertebra pattern
Thing, is used widely rapidly in fields such as developmental biology, toxicity of compound evaluation and human disease model researchs.
Zebrafish embryo after fertilization 14h (14hpf, 14hours post-fertilization) kidney germinates,
During 72hpf, kidney development completes.Early in 48hpf it is observed that glomerular filtration phenomenon.Brachydanio rerio kidney is divided into young stage
Pronephridiostome and the middle kidney of adult fish phase, pronephridiostome is made up of a pair of nephron, and glomerule is at embryo's median line of dorsal aorta veutro
Merge, renal tubules separation both sides.The anatomical structure of pronephridiostome is simpler than the middle kidney of adult fish and the metanephros of mammal, but in groups of cells
Similar to the kidney of mammal on one-tenth and molecular level, and possessed equally complicated biological function.Brachydanio rerio develops first
In week, because the gill is not also developed completely, rely primarily on kidney and discharge moisture unnecessary in vivo and metabolic waste, this stage is young in addition
Fish need not feeding can survive, and interference factor is few, is very beneficial for carrying out the related research of Toxicity of Kidney.Zebrafish embryo conduct
Whole animal model, can truly reflect pathological change under environmental activity in cardiovascular system, immunocyte etc. for the kidney, tool
For the high advantage of living animal experimental result reliability.
At present, carried out in compound Toxicity of Kidney research using zebrafish embryo, the detection to kidney injury is mainly wrapped
Include the several respects such as phenotypic alternation, Pathological and glomerular filtration rate estimation.Wherein, glomerular filtration rate is directly to reflect kidney
The important quantizating index of functional statuses, is to be surveyed by calculating the clearance rate of the similar substances such as internal inulin in zebrafish embryo
, need the inulin of revealing label through, in embryo's venae cordis hole injection blood circulation, because embryo is less, the method is to instrument
Higher with technical requirements, operation easier is big.Disease leads to body pathology physiology to change, and necessarily causes metabolism group in tissue
The change of point content, to these components, the inspection of especially more closely related with organ dysfunction state biomarker content
Survey, be diagnose the illness generation, the important evidence of development.Creatinine is the metabolite of muscle, is turned by precursor compound phosphagen
Change forms, and is mainly excreted by kidney.During impaired renal function, creatinine is accumulated in vivo becomes harmful toxins.Clinically, examine
Survey blood, urine creatine value is the important indicator understanding renal function.Zebrafish embryo small volume is it is difficult to obtain blood or urine sample
This, at present, creatinine level change aspect after data information about creatinine content in zebrafish embryo body and impaired renal function
Research still belongs to blank.In view of significance in the detection of kidney work(for the creatinine, carrying out compound kidney poison using zebrafish embryo
It is necessary to investigate to this index by accurate, the reliable method of one kind during Journal of Sex Research, kidney work(is improved with supplement further
Can appraisement system.
The method that the present invention utilizes Liquid Chromatography-Tandem Mass Spectrometry, surveys to the creatinine content in zebrafish embryo tissue
Fixed, and compare with the creatinine content value of normal zebrafish embryo tissue, for the Toxicity of Kidney of quick, reliable detection compound
Effect.
Content of the invention
The present invention is directed to the deficiencies in the prior art, provides a kind of detection zebra fish tissues creatinine content that passes through to evaluate compound
The method of Toxicity of Kidney.
It is an object of the invention to provide a kind of Toxicity of Kidney evaluation methodology as index for the creatinine content in organize, for
Toxicity of Kidney research, in the research carrying out compound Nephrotoxicity using via zebra fish embryo model, by embryonal tissue
In creatinine composition extracted, and measure its content using Liquid Chromatography-Tandem Mass Spectrometry method, can be with embryonal tissue
Creatinine content becomes the index being turned to detect renal function, sets up a kind of quick, accurate, compound Toxicity of Kidney of high specificity
Appraisement system.
Technical solution of the present invention is as follows:
A kind of by measure organize in creatinine changes of contents detection compound Toxicity of Kidney method, step is as follows:
(1) zebrafish embryo developed 3～6 days is moved in the culture water containing testing compound, at 28 DEG C, 14h light
According to cultivating 24～72h under/10h dark condition, as test group, then remove dead embryo, clean embryonic surface with pure water residual
After staying liquid, obtain embryo after cultivating, after described cultivation, embryo is less than 7 days away from fertilization time；
Inventor is found by research, fertilization time more than 7 days after embryo, due to being affected by multiple interference factors,
Detection accuracy can be led to reduce.
Under the same conditions with culture Aquaponic as fetal tissues group；
(2) to after cultivate, embryo adds inner mark solution, through homogenate, Multi-layer technology polar compound therein, extracts sample warp
After nitrogen dries up, add pure water dissolving, then cross 0.45 μm of polyether sulfone aqueous phase filter membrane, filtrate is injected lc-ms/ms, carry out separating
Analysis；Then respectively creatinine standard solution and internal standard standard solution are injected lc-ms/ms, examined under the same conditions
Survey；
(3) according to equation below:
cTesting sample=cStandard substance×aaTesting sample/aaStandard substance
In formula: c is to extract sample concentration, aa is chromatographic peak area；
Calculate the creatinine in testing sample and internal standard content, calculate the flesh in each group embryo samples further according to internal standard extraction ratio
Acid anhydride content；
(4) in embryonal tissue, creatinine content is represented with means standard deviation, using independent samples t test method com-parison and analysis group
Between difference significance, when in test group embryonal tissue creatinine content be higher than fetal tissues group embryonal tissue in creatinine content (p
< 0.05), and exceed the normal dividing value scope of corresponding development age embryonal tissue creatinine content, illustrate that embryo's renal function is damaged
Evil, test-compound has Toxicity of Kidney；
Randomly select different healthy Brachydanio rerio male and female parent fishs, pairing spawning in research, collect and obtain different developmental phases
Each 20 groups of zebrafish embryo, every group of 50 embryos, middle step (2) (3) (4) as stated above, extract detection tissue in flesh
Acid anhydride content, result is as follows:
Creatinine content (ng/50 embryo) in table 1 different developmental phases embryonal tissue
Single sample k-s check analyses show the equal Normal Distribution of above-mentioned each group of data, determine different growth instar embryo groups
The normal dividing value scope (95%) knitting middle creatinine content is:
Creatinine level in after fertilization 4d zebra fish tissues is 30.91～49.09ng/50 embryo；After fertilization 5d zebra
Creatinine level in fish tissues is 36.56～56.32ng/50 embryo；Creatinine level in after fertilization 6d zebra fish tissues is
58.38～82.06ng/50 embryo；Creatinine level in after fertilization 7d zebra fish tissues is 84.47～124.81ng/50
Embryo.
According to currently preferred, in described step (1), zebrafish embryo used is wild type or ab system Brachydanio rerio embryo
Tire.
According to the present invention it is further preferred that above-mentioned embryo be fertilization more than 3 days, kidney completed develop wild type or
Ab system zebrafish embryo.
According to currently preferred, in described step (1), the culture water containing testing compound is to add in culture water
Testing compound or the mixing of testing compound and cosolvent.
The purpose of above-mentioned cosolvent is to promote the dissolving of testing compound, and cosolvent is not right in concentration range planted agent used
Embryo produces Toxicity of Kidney effect.
The concentration of above-mentioned testing compound differs, this area according to the toxicity difference of different testing compound generations
Technical staff can voluntarily adjust selection according to experimental conditions.
According to the present invention it is further preferred that above-mentioned culture water component is:
Nacl5mmol/l, kcl0.17mmol/l, cacl20.4mmol/l, mgso40.16mmol/l, deionized water is prepared.
According to currently preferred, in described step (1), what during culture, every 24h changed 3/4 volume contains test compounds
The culture water of thing.
According to currently preferred, in described step (2), the internal standard substance adding in embryo samples is cimetidine standard
Product solution.Cimetidine structural formula is as follows:
Creatinine content in described embryonal tissue is the quality of contained creatinine in each fixed number purpose embryonal tissue.Creatinine
Structural formula is as follows:
Beneficial effect
1st, the present invention is extracted to the creatinine in zebrafish embryo tissue and is measured, and is turned to examine with the change of creatinine content
Survey the index of embryo's kidney functional statuses, compared with existing zebrafish embryo renal function detection side, not only achieve kidney
The quantitatively evaluating of function, and more representative and universality.
2nd, the present invention adopts the creatinine in liquid chromatography tandem mass spectrometry determination sample, makes full use of chromatographic column high efficiency separation
Efficiency, the optimization of chromatographic condition in addition, it is to avoid the measurement deviation that the interference component in tissue sample leads to, improve mensure knot
The accuracy of fruit and reliability；Simultaneously with cimetidine standard substance for internal standard analyte, effectively reduce what experimental implementation caused
Error.
3rd, model organism zebra fish embryo used in the present invention, both had the advantages that cell in vitro strain can rapid screening,
There is the advantage of checking in living animal body again, carry out the Toxicity of Kidney evaluation of large-scale compound using zebrafish embryo, have
Help improve conventional efficient and reduce experimental cost.
4th, after the present invention strictly limits cultivation, embryo is less than 7 days away from fertilization time, such that it is able to improve testing result
Accuracy.
Brief description
Fig. 1 is the photo of zebrafish embryo (5dpf)；
Wherein: Fig. 1 a is matched group zebrafish embryo (5dpf) photo；
Fig. 1 a ' is matched group zebrafish embryo (5dpf) tissue slice (transverse section, he dyes) photo；
Fig. 1 b is that zebrafish embryo (4dpf) processes the photo after 24h through Aristolochic Acid a (4 μ g/ml)；
Fig. 1 b ' is zebrafish embryo (4dpf) through Aristolochic Acid a (4 μ g/ml) process the tissue slice after 24h (transverse section,
He dyes) photo；
In figure: gl, glomerule；Arrow, near the eyes edema；*: glomerule cystic dilatation.
Fig. 2 is the creatinine content block diagram of variable concentrations Aristolochic Acid a treatment group zebrafish embryo tissue；
*: with matched group ratio, p < 0.05, * *: with matched group ratio, p < 0.01.
Fig. 3 is zebrafish embryo renal tissue section (he dyeing) photo；
Wherein: Fig. 3 a is matched group embryo (5dpf) renal tissue section (he dyeing) photo；
Fig. 3 b is zebrafish embryo (6dpf) renal tissue section (he dyeing) after 25 μm of ol/l notalin effect 48h
Photo；
In figure: gl: glomerule, pt: pronephric duct.*: cystic dilatation；
Fig. 4 is the creatinine content block diagram that 25 μm of ol/l notalins process zebrafish embryo (6dpf) tissue after 48h；
In figure: * *: with matched group ratio, p < 0.01.
Specific embodiment:
With reference to embodiment, technical scheme is further elaborated, but institute of the present invention protection domain is not limited to
This.
Laboratory animal: wild type used or ab system health Brachydanio rerio, are this area ordinary commercial products, at 28 DEG C, 14h light
Cultivate according under/10h dark condition, daily in 9:30 (am) and 4:30 (pm) feeding fairy shrimp twice.Adult fish is pressed by the ovulation day before yesterday
Sex ration 2:1 is put in spawning tank, middle placement dividing plate, is placed in dark surrounds, pumps dividing plate, light stimulation before next day bright light
So that it is ovulated, after half an hour, adult fish is pulled out, so that ovulation period is controlled within half an hour, to reduce the difference of development time between embryo
Different.Collect germ cell, rinsed after 3 times with new culture water, be placed in 28 DEG C of incubators, keep 14h illumination/10h dark cycle training
Support, middle every 24h exchanges treaties 1/3 water, and suctions out dead embryo in time.
Preparation of reagents: creatinine standard substance and cimetidine standard substance are purchased from Nat'l Pharmaceutical ＆ Biological Products Control Institute, creatinine
It is configured to 10 μ g/ml standard solutions with pure water dissolving, the dissolving of cimetidine pure water is configured to 4 μ g/ml standard solutions.
Culture water component is as follows:
Nacl5mmol/l, kcl0.17mmol/l, cacl20.4mmol/l, mgso40.16mmol/l, deionized water is prepared.
Embodiment 1: variable concentrations Aristolochic Acid a acts on to the Toxicity of Kidney of zebrafish embryo (4dpf)
Reagent: Aristolochic Acid a standard substance are purchased from National Institute for Food and Drugs Control, are prepared with dmso solution
5mg/ml storing liquid, 4 DEG C of preservations, the longest holding time is 1 month, is diluted to desired concn with culture water during experiment.
Selection standard specification 24 porocyte culture plates, every in the hole adds appropriate Aristolochic Acid a storing liquid, with culture water dilution
To 2ml, obtain 0.5 μ g/ml, 2 μ g/ml, 4 μ g/ml tri- concentration group.Matched group is the culture aqueous solution containing 0.5 ‰ dmso, body
Amass as 2ml.With suction pipe, all sample in the hole solution are carefully blown and beaten mixing, the random zebrafish embryo drawing 4 days sizes of growth, moves
Enter above-mentioned sample in the hole, every 15 embryos in hole, every group of 4 holes, all groups are all provided with three parallel repetitions.After adding a cover closing, put into 28
In DEG C incubator, 14h illumination/10h is dark lower to be incubated 24h.
Each group embryo after collection process, removes dead embryo, and makes embryo number between each group identical.By embryo's pure water rinsing
After 3 times, move in 1.5ml eppendorf pipe, exhaust moisture as far as possible, adds the pre- cold methanol of 400 μ l, 124 μ l precooling pure waters, 1 μ
L cimetidine inner mark solution, is homogenized 2min, then adds 400 μ l chloroforms into every pipe, vortex oscillation 60s, stratification, so
4 DEG C afterwards, under the conditions of 10000 × g, it is centrifuged 5min, careful supernatant liquid of drawing moves to new 1.5ml eppendorf pipe, and nitrogen dries up,
Dry up sample and be put in -70 DEG C of preservations.
Using shimadzu vp-ods post (250mm × 4.6mm, 3 μm), analysis time 12min, sample size 5 μ l.Eluting
Condition: mobile phase a (methanol) and mobile phase b (pure water) carry out gradient elution (being shown in Table 2) according to different proportion, flow rate of mobile phase:
1.0ml/min.
Mass Spectrometry Conditions: electric spray ion source polarity: spray voltage: 3000v；Gasification temperature: 350 DEG C；Sheath air pressure: 60arb；
Assist gas pressure: 30arb；Ion transfer tube temperature: 300 DEG C；Collision gas (ar) 1.5mtorr；Scan pattern srm.
Table 2 utilizes flow visualizing used by creatinine in lc-ms/ms detection zebrafish embryo tissue
Taking nitrogen to dry up sample adds 1ml pure water fully to dissolve, and crosses 0.45 μm of polyether sulfone aqueous phase filter membrane, takes 5 μ l filtrate injections
Lc-ms/ms is carried out analyzing by above-mentioned condition.
Respectively creatinine standard solution and internal standard standard solution are diluted that (creatinine standard substance are diluted to concentration
100ng/ml, internal standard standard substance are diluted to concentration 15ng/ml), respectively take 5 μ l injection lc-ms/ms, under identical chromatographic condition
It is measured, calculate creatinine and the internal standard content in testing sample using external standard method, and calculated in sample according to internal standard extraction ratio
Creatinine content；Computing formula:
cTesting sample=cStandard substance×aaTesting sample/aaStandard substance
C: extract sample concentration, aa: chromatographic peak area.
Result shows, 4dpf zebrafish embryo is contaminated through Aristolochic Acid and processed after 24h, and creatinine content significantly raises, 0.5 μ
After g/ml, 2 μ g/ml, 4 μ g/ml Aristolochic Acid a is processed, embryo's creatinine content value is respectively 55.79 ± 5.4,72.91 ± 8.1,
71.60 ± 8.3ng/50 embryo, higher than matched group (44.7 ± 7.6ng/50 embryo) (table 3), and have significant difference (p <
0.05) (as shown in Figure 2).
The rising of embryo's creatinine value illustrates that Aristolochic Acid a causes the damage of zebrafish embryo renal function after processing, and kidney is little
Ball filtering function declines, and leads to creatinine to be accumulated in vivo.Microscopic observation, typical edema oculi in Aristolochic Acid a treatment group embryo
Performance (as shown in Figure 1), kidney function suffers damage and causes tissue edema.By embryo through fixation, paraffin embedding, do pathology
Sections observation, finds that short texture, cell arrangement disorder and Cystic changes in Aristolochic Acid a treatment group embryo's renal glomerulus
(as shown in Figure 1), shows that renal tissue structure suffers damage.
Table 3 lc-ms/ms detects the creatinine content in variable concentrations Aristolochic Acid a treatment group zebrafish embryo
N: every group Duplicate Samples number；Aa: chromatographic peak area；*: with matched group ratio, p < 0.05, * *: with matched group ratio, p <
0.01.
Clinically, the Chinese crude drug taking the composition of a containing Aristolochic Acid can seriously damage human kidney function, also sends out in research
Existing Aristolochic Acid a has development toxicity (yu-ju ding, yau-hung chen.toxicology and to Brachydanio rerio kidney
Applied pharmacology261 (2012): 59 65.), in the method, in embryonal tissue after processing through Aristolochic Acid a
Creatinine content value raise, and exceed accordingly normal dividing value scope, cut into slices performance in conjunction with renal tissue, show that Aristolochic Acid a has
There is Toxicity of Kidney to act on, also demonstrate that the testing result of this method is accurate simultaneously.
Embodiment 2: the toxic action to zebrafish embryo (4dpf) kidney for the notalin
Reagent: notalin standard substance are purchased from Nat'l Pharmaceutical ＆ Biological Products Control Institute, with the culture water containing 25% (v/v) ethanol
Solution solution prepares 10mm liquid storage, 4 DEG C of preservations, is diluted to desired concn with culture water during experiment.
Selection standard specification 6 porocyte culture plates, every in the hole adds appropriate notalin liquid storage, is diluted to 25 μ with culture water
Mol/l concentration level, every hole final volume is 6ml.Matched group is the culture aqueous solution containing isoconcentration ethanol, and volume is 6ml.With inhaling
All sample in the hole solution are carefully blown and beaten mixing by pipe, the random zebrafish embryo drawing 4 days sizes of growth, move into above-mentioned sample hole
Interior, every 60 embryos in hole, every group sets three parallel multiple holes.After adding a cover closing, put in 28 DEG C of incubators, 14h illumination/10h is black
Secretly lower continuation incubation 48h, solution of middle replacing.
Each group embryo after collection process, removes dead embryo, and makes embryo number between each group identical.By embryo's pure water rinsing
After 5 times, move in 1.5ml eppendorf pipe, exhaust moisture as far as possible, adds the pre- cold methanol of 400 μ l, 124 μ l precooling pure waters, 1 μ
L cimetidine inner mark solution, is homogenized 2min, then adds 400 μ l chloroforms into every pipe, vortex oscillation 60s, stratification, so
4 DEG C afterwards, under the conditions of 10000 × g, it is centrifuged 5min, careful supernatant liquid of drawing moves to new 1.5ml eppendorf pipe, and nitrogen dries up,
Dry up sample and be put in -70 DEG C of preservations.
Using shimadzu vp-ods post (250mm × 4.6mm, 3 μm), analysis time 12min, sample size 5 μ l.Eluting
Condition: mobile phase a (methanol) and mobile phase b (pure water) carry out gradient elution according to different proportion, condition is as shown in table 2, mobile phase
Flow velocity: 1.0ml/min.Mass Spectrometry Conditions: electric spray ion source polarity: spray voltage: 3000v；Gasification temperature: 350 DEG C；Sheath gas
Pressure: 60arb；Assist gas pressure: 30arb；Ion transfer tube temperature: 300 DEG C；Collision gas (ar) 1.5mtorr；Scan pattern
srm.
Taking nitrogen to dry up sample adds 1ml pure water fully to dissolve, and crosses 0.45 μm of polyether sulfone aqueous phase filter membrane, takes 5 μ l filtrate injections
Lc-ms/ms is carried out analyzing by above-mentioned condition.
Respectively creatinine standard solution and internal standard standard solution are diluted (creatinine standard substance are diluted to concentration:
100ng/ml, internal standard standard substance are diluted to concentration: 15ng/ml), respectively take 5 μ l injection lc-ms/ms, under identical chromatographic condition
It is measured, calculate creatinine and the internal standard content in testing sample using external standard method, and calculated in sample according to internal standard extraction ratio
Creatinine content；Computing formula:
cTesting sample=cStandard substance×aaTesting sample/aaStandard substance
C: extract sample concentration, aa: chromatographic peak area
Result shows, 4dpf zebrafish embryo is processed after 48h through each concentration group notalin, and Microscopic observation embryo does not occur bright
Aobvious paramophia.Collect normal group and treatment group embryo, homogenate is extracted, sample will be extracted and introduce lc-ms/ms, and record 25 μm of ol/
The creatinine content of l notalin treatment group embryo is 88.0 ± 4.6ng/50 embryo, higher than Normal group 69.2 ± 6.5ng/
50 embryos (p < 0.01) (table 4) (as shown in Figure 4), and the creatinine normal dividing value scope more than 6d zebrafish embryo tissue.
Normal group and treatment group embryo are done with pathological section and he dyeing, shows 25 μm of ol/l notalin treatment group embryo kidneys
Dirty the performance such as glomerule cystis degeneration, the loosely organized, arrangement disorder of pronephric duct epithelial cell (as shown in Figure 3), and embryo is described
The structure of kidney and function are destroyed, and notalin has obvious Nephrotoxicity to zebrafish embryo.
Creatinine content in zebrafish embryo after table 4 lc-ms/ms detection notalin process
N: every group Duplicate Samples number；Aa: chromatographic peak area；*: with matched group ratio, p < 0.01.
In wu et al. (ting-shuan wu, jiann-jou yang, feng-yih yu et al.food and
chemical toxicology50(2012)4398–4404.) research in, 24hpf zebrafish embryo through notalin process after,
Renal function is severely damaged, and pronephridiostome and the change of pronephric duct organizational structure, this testing result one with this method
Cause, also demonstrate that the Toxicity of Kidney by the accurate detection compound of this method energy.
Claims (4)
1. a kind of method by measuring the changes of contents detection compound Toxicity of Kidney of creatinine in zebrafish embryo tissue, it is special
Levy and be, step is as follows:
(1) zebrafish embryo developed 3～6 days is moved in the culture water containing testing compound, at 28 DEG C, 14 h illumination/
Cultivate 24～72h under 10 h dark conditions, as test group, remove dead embryo, clean through pure water after embryonic surface debris,
Obtain embryo after cultivating, after described cultivation, embryo is less than 7 days away from fertilization time；
Under the same conditions with culture Aquaponic as fetal tissues group；
The described culture water containing testing compound is to add testing compound or testing compound and cosolvent in culture water
Mixing；
Above-mentioned culture water component is:
Nacl 5mmol/l, kcl 0.17mmol/l, cacl20.4mmol/l, mgso40.16mmol/l, deionized water is prepared；
(2) to after cultivate, embryo adds inner mark solution, through homogenate, Multi-layer technology polar compound therein, extracts sample and blows through nitrogen
After dry, add pure water dissolving, then cross 0.45 μm of polyether sulfone aqueous phase filter membrane, filtrate is injected lc-ms/ms, carry out separation point
Analysis；Then respectively creatinine standard solution and internal standard standard solution are injected lc-ms/ms, detected under the same conditions；
The internal standard substance adding in embryo samples is cimetidine standard solution；
(3) according to equation below:
cTesting sample= cStandard substance×aaTesting sample/aaStandard substance
In formula: c is to extract sample concentration, aa is chromatographic peak area；
Calculate the creatinine in testing sample and internal standard content, calculate the creatinine in each group embryo samples further according to internal standard extraction ratio and contain
Amount；
(4) in embryonal tissue, creatinine content is represented with means standard deviation, using independent sampletBetween method of inspection com-parison and analysis group
The significance of difference, under the level of p < 0.05 significance, when in test group embryonal tissue, creatinine content is higher than fetal tissues group
Creatinine content in embryonal tissue, and exceed the normal dividing value scope of corresponding development age embryonal tissue creatinine content, embryo is described
Renal function suffers damage, and test-compound has Toxicity of Kidney；
In above-mentioned each development age embryonal tissue, the normal dividing value scope of creatinine content is:
Creatinine level in after fertilization 4d zebra fish tissues is 30.91～49.09 ng/50 embryos；After fertilization 5d Brachydanio rerio
Creatinine level in tissue is 36.56～56.32 ng/50 embryos；Creatinine level in after fertilization 6 d zebra fish tissues is
58.38～82.06 ng/50 embryos；Creatinine level in after fertilization 7d zebra fish tissues is 84.47～124.81ng/50
Embryo.
2. the method for claim 1 is it is characterised in that in described step (1), zebrafish embryo used is wild type
Or ab system zebrafish embryo.
3. it is characterised in that above-mentioned zebrafish embryo is fertilization more than 3 days, kidney completes method as claimed in claim 2
The wild type developed or ab system zebrafish embryo.
4. the method for claim 1 is it is characterised in that in described step (1), during culture, every 24h changes 3/4 volume
The culture water containing testing compound.
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| CN105169415B (en) * | 2015-08-10 | 2018-07-24 | 山东省科学院生物研究所 | A method of screening has protection zebra fish liver function reactive compound |
| CN105709242B (en) * | 2016-05-03 | 2018-10-26 | 山东省科学院生物研究所 | A method of zebra fish gastrointestinal tract is marked using calcein |
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