CN108251508A - The method for assessing the neurotoxicity of water pollutant - Google Patents
The method for assessing the neurotoxicity of water pollutant Download PDFInfo
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- CN108251508A CN108251508A CN201810084597.0A CN201810084597A CN108251508A CN 108251508 A CN108251508 A CN 108251508A CN 201810084597 A CN201810084597 A CN 201810084597A CN 108251508 A CN108251508 A CN 108251508A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The method that the present invention provides the neurotoxicity of assessment water pollutant.Including：Prepare the decis solution of 10% earth's surface water concentration；The continuous exposure that suitable zebra fish carries out seven days by a definite date in certain pollutant solution of water aeration and 10% earth's surface water concentration respectively is chosen, respectively as blank group and experimental group；The zebra fish brain of above-mentioned blank group and experimental group is dissected, extraction total serum IgE, reverse transcription carry out real-time fluorescence quantitative PCR amplification into cDNA；With 2- △ △ CTFor method to carrying out relative quantitative assay, and draw line chart to experimental data, assessing water pollutant according to experimental result may be to the influence of the function of M-ChR (M1 receptors).Step is simple and convenient to operate, is highly practical.
Description
Technical field
The invention belongs to environmental monitoring, it is related to a kind of assessing water pollution to health using molecular engineering means
The method of potential hazard.
Background technology
With the completion that zebra fish gene order-checking works, it is found that the conservative degree of the gene of zebra fish and the gene of the mankind is high
Up to 85%, this causes zebra fish to become a kind of model animal for studying human diseases, has been to be concerned by more and more people.Meanwhile
Gradually establishing and improving for Zebrafish Embryo genetics technology is also provided for the human disease model's of zebra fish with analysis
Good basis.Decis has selection toxicity, compared to birds and mammality etc., fish to the toxicity of decis more
It is sensitive.Thus, it is a good selection as monitoring object to select zebra fish.
At present, the method for Water Quality Evaluation is various, such as physico-chemical analysis method (such as titration, spectrophotometric analysis), modern instrument
Device analysis method (chromatography, spectrum analysis etc.), biotest methodology etc..And the life level being related to is also than wide, from
The research that group arrives bion to population again can be found everywhere.And change of water quality is assessed in molecular level, it is relatively deficient.Value
It obtains it is noted that the poisonous effect of water pollutant, what is had can show at once, and some incubation periods are relatively long.This is just needed
It will be from the variation of the internal physiological reacting condition water quality of aquatile.
Patent CN201510315920.7 discloses a kind of double crush syndrome quantitative detecting method, passes through double antibodies sandwich
The content of standard measure detection zebra fish M3a receptor proteins and the variation for assessing water quality.But due to double-antibody method detection time compared with
It is long, be difficult to it is timely, accurate.
The intelligent diligent paper of history《Drug neurotoxicity evaluates the foundation of zebra fish model and ketamine neurodevelopment toxic mechanism
Research》The zebra fish nervous centralis nmda receptor subunit expression of flax dose ketamine is detected using RT-PCR methods, to comment
Estimate zebra fish juvenile fish neurodevelopment toxic effect and dosage-behavior effect mechanism.But due to nmda receptor subunit content, distribution model
Enclose relatively narrow, shortage wide applicability.
Invention content
In order to overcome the above problem, the application is directed in existing Water Quality Evaluation, about decis methods of Toxicity Assessment
It is insufficient, it is proposed that a kind of relative expression using M1 receptors assesses water pollutant (decis toxicity) to human health
The method of potential hazard.
M1 receptors are a hypotypes of mAChR, are distributed in the brain the most extensively, account for brain skin
Layer and the 40%-50% of hippocampus.M1 receptors Ahl tribulus sea silent sickness (AD) is in close relations.4 amyloid deposit is alzheimer '
One of feature of silent disease, the activation of M1 receptors can protect neuron from 4 amyloid (Ah) toxicity.M1 receptors and cognitive process
It is related, participate in adjusting learning and memory, protect cells from damaging caused by apoptotic effect, including DNA damage, oxidative stress and
The damage of neuron linear plastochondria in cortex.Also containing M1 receptors in cardiac muscle cell, and M1 receptors can lead to the decline of heart rate.It utilizes
The substantial connection of M1 receptors and neurogenic disease.
To achieve these goals, the present invention adopts the following technical scheme that：
A kind of method assessed using zebra fish the potential risk of the neurotoxicity of water pollutant, including：
Prepare the decis solution of 10% earth's surface water concentration；
Suitable zebra fish is chosen to be scheduled to last in certain pollutant solution of water aeration and 10% earth's surface water concentration respectively
The continuous exposure of seven days, respectively as blank group and experimental group；
The zebra fish brain of above-mentioned blank group and experimental group is dissected, extraction total serum IgE, reverse transcription are gone forward side by side into cDNA
Row real-time fluorescence quantitative PCR expands；
With 2- △ △ CTMethod assesses water to carrying out relative quantitative assay, and draw line chart to experimental data, according to experimental result
Middle pollutant may be to the influence of the function of M-ChR (M1 receptors).
In order to ensure to the accuracy of decis toxicity evaluation in water, quick, it is necessary to find relationship between expression and bromine cyanogen chrysanthemum
The closely related receptor of ester content, meanwhile, this receptor must also have the characteristics of rich content, widely distributed and vdiverse in function,
To ensure the broad applicability of this method.For zebra fish, although 5 kinds of receptor subtypes (M1-M5) have expression in the brain,
But M1 contents are most abundant, account for cerebral cortex and the 40%-50% of hippocampus, it is often more important that, M1 receptor Ahl tribulus sea silent sickness
(AD) in close relations, 4 amyloid deposit is one of feature of Alzheimer disease, and the activation of M1 receptors can protect neuron
From 4 amyloid (Ah) toxicity, decis toxicity in water preferably can be selectively assessed.In addition, in addition to central nervous system
And peripheral nervous system, M1 receptors are also distributed widely in heart, smooth muscle etc., convenient for detaching and extracting.Therefore, this in research
Application preferably carries out RT-PCR method detections using M1 receptors.
Experimental study further demonstrates that：For zebra fish, M1 receptor relative expression quantities under different decis concentration
Trend generally conform to sine of the third order function f (x)=a1*sin(b1*x+c1)+a2*sin(b2*x+c2)+a3*sin(b3*x+c3),
Both can preferably assess the time that zebra fish is influenced by decis, be practical polluted water region in pollution sources judgement and divide
Analysis provides effectively data supporting；Again can by the fitting of the trend to M1 receptor relative expression quantities, judge in pollution sources whether
There are the pollutants of other non-decis, improve the anti-interference of testing result.
On the other hand, the method for the present invention is from receptor oneself expression, and monitoring object is receptor rather than receptor protein, phase
Than the amount in double-antibody method with the indirect characterization receptor of receptor protein amount, with more direct, accuracy；In experimental implementation side
Face needs the preparation of standard items curve in double-antibody method, takes consumptive material, and the present invention does not need to the preparation of standard items curve,
Be to using depending on expression of receptor cq values carry out relative quantitative assay, this method have it is easy to operate, time saving and energy saving, consumptive material is few
Feature；In terms of experiment condition, double-antibody method is more harsh to the requirement for being coated antibody, and the uncertainty of experiment is larger
(coated antibody requirement purity is high, and between 9.0-9.6,4 DEG C are placed for 24 hours pH value.Coated optimum concentration and enzyme labelled antibody are most
Suitable working concentration is both needed to be determined by titration experiments), compared to double-antibody method, this experiment is with more scientific, preciseness.
Preferably, the preparation method of the decis solution is：0.02g decis is dissolved in 5mLDMSO, is added in
995mL deionized waters are configured to 1L mother liquors (0.02g/L)；100 μ L mother liquors are taken in conical flask, with deionized water constant volume to 1L,
It is made into the experiment solution of a concentration of 0.002mg/L of 1L.
Preferably, the concentration of the DMSO in water is less than 0.5wt%.
Preferably, it is described dissection processing the specific steps are：Zebra fish is taken, is put into 1.5mL EP pipes (eppendorf pipes)
In, carry out asphyxia processing；The brain of zebra fish is dissected under aseptic technique.
Preferably, the experimental condition of the zebra fish is：22 ± 2 DEG C of temperature；Photoperiod:16h illumination, 04:00-20:00,
Intensity of illumination 4000lx；8h is dark, and 20:00-04:00；Daily 8:30 and 16:30 each feedings are primary.
Preferably, the stem length 3cm of the zebra fish, tail length 2cm.
In order to ensure the Stability and veracity of testing result, the application to the PCR detection methods of zebra fish M1 receptors into
Optimization is gone, it is preferred that the specific method of the PCR amplification is：
Using two-step method, specific amplification condition is as follows：
Table 1
Preferably, the kit of the extraction total serum IgE is total RNA from animal tissues extracts kit DP431.
Preferably, the reverse transcription is into the model KR106FastQuant RT Kit of the kit of cDNA.
Preferably, the reagent of the real-time fluorescence quantitative PCR is SuperReal PreMix Plus (SYBR Green)
(FP205).Beneficial effects of the present invention
(1) M-ChR is the receptor that neurotransmitter-acetylcholine is specifically bound, and is widely present in parasympathetic
On the effector cell that postganglionic fibre dominates.After acetylcholine is combined with this receptoroid, a series of parasympathetics can be generated
Nerve endings excitedly effect.By the relative expression of M1 receptors, the decis of 10% earth's surface water concentration is analyzed to zebra fish
The influence of brain M1 receptors.Using this model organism of zebra fish, by taking decis this pollutant as an example, from molecular level pair
The potential risk of the neurotoxicity of water pollutant is assessed, to improve Water Quality Evaluation system.
(2) appraisal procedure of the present invention is simple, efficient, highly practical, easy to spread.
Description of the drawings
The accompanying drawings which form a part of this application are used for providing further understanding of the present application, and the application's shows
Meaning property embodiment and its explanation do not form the improper restriction to the application for explaining the application.
Fig. 1 is the photo of zebra fish；
Fig. 2 is zebra fish cyclic culture device；
Fig. 3 is experiment instrument；
Fig. 4 is the variation of zebra fish brain M1 relative expression quantities under 0.52 μ g/L concentration；
Fig. 5 is the variation of zebra fish brain M1 receptor relative expression quantities under 2 μ g/L concentration.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.It is unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
The potential risk of the neurotoxicity of water pollutant is assessed using zebra fish, is included the following steps：
1. the selection of biological subject：(22 ± 2 DEG C of temperature is randomly selected under experimental condition；Photoperiod:16h illumination, 04:00-
20:00, intensity of illumination 4000lx；8h is dark, and 20:00-04:00；Daily 8:30 and 16:30 each feedings are primary) adult zebra
Fish (stem length 3cm, tail length 2cm) is several to be tested, and it is 1 to make female-male proportion:1, stop feeding for 24 hours before experiment.
2. the preparation of contaminant trace species solution：According to People's Republic of China's water environment quality standard, experiment is determined
With the concentration of drug, make its 10% for earth's surface water concentration.0.02g decis is dissolved in 5mL DMSO, 995mL is added in and goes
Ionized water is configured to 1L mother liquors (0.02g/L).100 μ L mother liquors is taken, with deionized water constant volume to 1L, to be made into 1L in conical flask
A concentration of 0.002mg/L experiment solution.That is, the decis solution of 10% earth's surface water concentration (0.002mg/L).
Pay attention to：It is dissolved using the DMSO of appropriate volume, the concentration of DMSO in water is made to be less than 0.5%, to ensure
DMSO will not interfere experiment.DMSO is a kind of water-soluble compound, can dissolve most organic compounds, even
Inorganic salts can also be dissolved.It the advantage is that toxicity is extremely low, select DMSO that can promote trace to the maximum extent as organic solvent
The dissolving of pollutant, and interference of the organic solvent to experimental result is avoided to the maximum extent.
3. it is tested using fluorescent quantitative PCR technique：
(1) exposure experiment and sampling：
1) suitable zebra fish is chosen respectively in certain pollutant solution of water aeration and 10% earth's surface water concentration into behavior
The continuous exposure of seven days phases；
2) it samples：Since exposed 0h, operation is sampled every 4 hours.Choose the tissue conduct of 2 zebra fish
One sample (i.e. each parallel two brains), forms a parallel control, every group of three parallel controls.It is as follows：
Table 2
3) processing of sample：Each time point takes out 6 zebra fish, is put into 1.5mLEP pipes, carries out asphyxia processing.
The brain of zebra fish is dissected under aseptic technique, each two brain tissue has three as a sample, each time point
A sample, i.e. three parallel groups；
4) into cDNA and real time fluorescent quantitative is carried out to extraction, the reverse transcription of brain tissue progress total serum IgE using kit
PCR is operated, and records experimental data.
4. the processing and interpretation of result of data：
With 2- △ △ CTMethod carries out relative quantitative assay to experimental data, by way of drawing line chart, observes blank group
With the trend of experimental group M1 receptors variation.And be compared the line chart of blank group and experimental group, analysis decis is to spot
The influence of horse fish M1 expression of receptor.Assessing water pollutant according to experimental result may be to the function of M-ChR (M1 receptors)
Influence.
Embodiment 1
First, the selection of biological subject
(22 ± 2 DEG C of temperature is randomly selected under experimental condition；Photoperiod:16h illumination, 04:00-20:00, intensity of illumination
4000lx；8h is dark, and 20:00-04:00；Daily 8:30 and 16:30 each feedings are primary) Adult Zebrafish (stem length 3cm, tail
Long 2cm) it is several tested, make female-male proportion be 1:1, stop feeding for 24 hours before experiment.
2nd, the preparation of contaminant trace species solution
Decis is pyrethroid insecticides, is current insecticide most widely used in the world, is had certain
Neurotoxicity.
According to People's Republic of China's water environment quality standard, the concentration of experimental chemical is determined, it is earth's surface to make it
The 10% of water concentration.0.02g decis is dissolved in 5mLDMSO, 995mL deionized waters is added in, is configured to 1L mother liquors
(0.02g/L).100 μ L mother liquors is taken, with deionized water constant volume to 1L, to be made into a concentration of 0.002mg/L's of 1L in conical flask
Experiment solution.That is, the decis solution of 10% earth's surface water concentration (0.002mg/L).
Pay attention to：It is dissolved using the DMSO of appropriate volume, the concentration of DMSO in water is made to be less than 0.5%, to ensure
DMSO will not interfere experiment.DMSO is a kind of water-soluble compound, can dissolve most organic compounds, even
Inorganic salts can also be dissolved.It the advantage is that toxicity is extremely low, select DMSO that can promote trace to the maximum extent as organic solvent
The dissolving of pollutant, and interference of the organic solvent to experimental result is avoided to the maximum extent.
3rd, it is tested using fluorescent quantitative PCR technique：
1st, exposure experiment and sampling：
(1) suitable zebra fish is chosen respectively in the decis solution of water aeration and 10% earth's surface water concentration into behavior
The continuous exposure of seven days phases；
(2) it samples：Since exposed 0h, operation is sampled every 4 hours.The tissue for choosing 2 zebra fish is made
For a sample (i.e. each parallel two brains), a parallel control, every group of three parallel controls are formed.It is as follows：
Table 3
(3) processing of sample：Each time point takes out 6 zebra fish, is put into 1.5mLEP pipes, carries out asphyxia processing.
The brain of zebra fish is dissected under aseptic technique, each two brain tissue has three as a sample, each time point
A sample, i.e. three parallel groups；
(4) extraction (DP431, Tiangeng) of total serum IgE, reverse transcription are carried out to brain tissue using kit into cDNA (KR106-
02, Tiangeng) and real-time fluorescence quantitative PCR operation (FP205-02, Tiangeng) is carried out, record experimental data.
The PCR amplification use two-step method, specific amplification condition, as shown in table 1.
4th, the processing and interpretation of result of data：
With 2- △ △ CTMethod carries out relative quantitative assay to experimental data, by way of drawing line chart, observes blank group
With the trend of experimental group M1 receptors variation.And be compared the line chart of blank group and experimental group, analysis decis is to spot
The influence of horse fish M1 expression of receptor.Assessing water pollutant according to experimental result may be to the function of M-ChR (M1 receptors)
Influence.
5th, it summarizes：
M-ChR is the receptor that neurotransmitter-acetylcholine is specifically bound, and is widely present in parasympathetic god
On the effector cell dominated through postganglionic fibers.After acetylcholine is combined with this receptoroid, a series of parasympathetic god can be generated
Through tip excitedly effect.By the relative expression of M1 receptors, the decis of 10% earth's surface water concentration is analyzed to zebra fish brain
The influence of portion's M1 receptors.Using this model organism of zebra fish, by taking decis this pollutant as an example, from molecular level to water
The potential risk of the neurotoxicity of middle pollutant is assessed, to improve Water Quality Evaluation system.
Embodiment 2
Culture and detection method are all same as Example 1, and the difference lies in a concentration of 0.25 μ g/L of decis.
Embodiment 3
Culture and detection method are all same as Example 1, and the difference lies in a concentration of 0.52 μ g/L of decis.
Embodiment 4
Culture and detection method are all same as Example 1, and the difference lies in a concentration of 0.75 μ g/L of decis.
Embodiment 5
Culture and detection method are all same as Example 1, and the difference lies in a concentration of 1.00 μ g/L of decis.
Embodiment 6
Culture and detection method are all same as Example 1, and the difference lies in a concentration of 1.52 μ g/L of decis.
Embodiment 7
Culture and detection method are all same as Example 1, and the difference lies in a concentration of 2.52 μ g/L of decis.
Embodiment 8
Culture and detection method are all same as Example 1, and the difference lies in a concentration of 3.00 μ g/L of decis.
Embodiment 9
Culture and detection method are all same as Example 1, and the difference lies in a concentration of 3.52 μ g/L of decis.
Embodiment 10
Culture and detection method are all same as Example 1, and the difference lies in a concentration of 4.00 μ g/L of decis.
Embodiment 11
Culture and detection method are all same as Example 1, and the difference lies in a concentration of 4.52 μ g/L of decis.
Embodiment 12
Culture and detection method are all same as Example 1, and the difference lies in a concentration of 5.00 μ g/L of decis.
With data instance a few days ago, we are based respectively on the data of 0.52 μ g/L and 2 μ g/L acquisitions as shown in Figure 4,5,
Of short duration expression quantity raising will be shown under pollutant stimulation, and is gradually restored to a slightly above normal level, is protected
Hold the balance of homeostasis of the organism in pollutant water body.Although various concentration (0.25~5 μ g/L dozens ofs concentration gradient)
The trend of lower M1 receptor relative expression quantities is not quite similar, but passes through MATLAB fittings and find that all trend generally conform to three
Rank SIN function f (x)=a1*sin(b1*x+c1)+a2*sin(b2*x+c2)+a3*sin(b3*x+c3)。
By establishing matched curve, the time that zebra fish is influenced by decis both can be preferably assessed, is practical
In polluted water region pollution sources judgement and analysis effectively data supporting is provided；Further through the trend to M1 receptor relative expression quantities
Fitting, judge to whether there is the pollutant of other non-decis in pollution sources, improve the anti-interference of testing result.
Finally it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not limited to this hair
It is bright, although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still
It can modify to the technical solution recorded in previous embodiment or equivalent replacement is carried out to which part.It is all in this hair
Within bright spirit and principle, any modification, equivalent replacement, improvement and so on should be included in protection scope of the present invention
Within.Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not to the scope of the present invention
Limitation, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art are not required to
Make the creative labor the various modifications or changes that can be made still within protection scope of the present invention.
Claims (10)
1. a kind of method assessed using zebra fish the potential risk of the neurotoxicity of water pollutant, feature are existed
In, including：
Prepare the decis solution of 10% earth's surface water concentration；
Suitable zebra fish is chosen to carry out in certain pollutant solution of water aeration and 10% earth's surface water concentration seven days by a definite date respectively
Continuous exposure, as blank group and experimental group respectively；
The zebra fish brain of above-mentioned blank group and experimental group is dissected, extraction total serum IgE, reverse transcription carry out reality into cDNA
When fluorescent quantitative PCR；
With 2- △ △ CTMethod is assessed dirty in water carrying out relative quantitative assay, and draw line chart to experimental data according to experimental result
Contaminating object may be to the influence of the function of M-ChR (M1 receptors).
2. the method as described in claim 1, which is characterized in that the preparation method of the decis solution is：By 0.02g bromines
Cyano chrysanthemate is dissolved in 5mLDMSO, is added in 995mL deionized waters, is configured to 1L mother liquors (0.02g/L)；100 μ L mother liquors are taken in taper
In bottle, with deionized water constant volume to 1L, it is made into the experiment solution of a concentration of 0.002mg/L of 1L.
3. method as claimed in claim 2, which is characterized in that the concentration of the DMSO in water is less than 0.5%.
4. the method as described in claim 1, which is characterized in that it is described dissection processing the specific steps are：Zebra fish is taken, is put into
In 1.5mL EP pipes, asphyxia processing is carried out；The brain of zebra fish is dissected under aseptic technique.
5. the method as described in claim 1, which is characterized in that the experimental condition of the zebra fish is：22 ± 2 DEG C of temperature；Light
Period:16h illumination, 04:00-20:00, intensity of illumination 4000lx；8h is dark, and 20:00-04:00；Daily 8:30 and 16:30 is each
Feeding is primary.
6. the method as described in claim 1, which is characterized in that the stem length 3cm of the zebra fish, tail length 2cm.
7. the method as described in claim 1, which is characterized in that the PCR amplification uses two-step method, specific amplification condition such as table
Shown in 1.
8. the method as described in claim 1, which is characterized in that the kit of the extraction total serum IgE is carried for total RNA from animal tissues
Take kit DP431.
9. the method as described in claim 1, which is characterized in that the reverse transcription is into the model KR106 of the kit of cDNA
FastQuant RT Kit。
10. the method as described in claim 1, which is characterized in that the reagent of the real-time fluorescence quantitative PCR is SuperReal
PreMix Plus(SYBR Green)(FP205)。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109633140A (en) * | 2018-12-19 | 2019-04-16 | 中国环境科学研究院 | A method of perfluorochemical neurodevelopment toxicity is evaluated using zebra fish |
| CN112136765A (en) * | 2020-09-29 | 2020-12-29 | 河南省农业科学院畜牧兽医研究所 | Pig ovary oxidative stress model and construction method and application thereof |
| CN113035356A (en) * | 2021-03-03 | 2021-06-25 | 首都医科大学 | Evaluation method for damage of cardiovascular and nervous systems by atmospheric particulates |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109633140A (en) * | 2018-12-19 | 2019-04-16 | 中国环境科学研究院 | A method of perfluorochemical neurodevelopment toxicity is evaluated using zebra fish |
| CN109633140B (en) * | 2018-12-19 | 2020-06-26 | 中国环境科学研究院 | Method for evaluating neurodevelopment toxicity of perfluorinated compounds by using zebra fish |
| CN112136765A (en) * | 2020-09-29 | 2020-12-29 | 河南省农业科学院畜牧兽医研究所 | Pig ovary oxidative stress model and construction method and application thereof |
| CN112136765B (en) * | 2020-09-29 | 2022-03-29 | 河南省农业科学院畜牧兽医研究所 | Pig ovary oxidative stress model and construction method and application thereof |
| CN113035356A (en) * | 2021-03-03 | 2021-06-25 | 首都医科大学 | Evaluation method for damage of cardiovascular and nervous systems by atmospheric particulates |
| CN113035356B (en) * | 2021-03-03 | 2023-12-08 | 首都医科大学 | Method for evaluating damage of atmospheric particulates to cardiovascular and nervous system |
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