CN102346147A - Method for detecting difference of cell toxicity between atmospheric nano particles and industrial nano particles - Google Patents
Method for detecting difference of cell toxicity between atmospheric nano particles and industrial nano particles Download PDFInfo
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- CN102346147A CN102346147A CN2011102738755A CN201110273875A CN102346147A CN 102346147 A CN102346147 A CN 102346147A CN 2011102738755 A CN2011102738755 A CN 2011102738755A CN 201110273875 A CN201110273875 A CN 201110273875A CN 102346147 A CN102346147 A CN 102346147A
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Abstract
The invention relates to a method for detecting the difference of cell toxicity between atmospheric nano particles and industrial nano particles. The method comprises the following steps of: a, using a culture medium to prepare a cell suspension, inoculating the cell suspension in a cell culture dish and culturing the culture dish in an incubator for 24 hours; b, mixing a prepared particulate matter solution and the culture medium and preparing into a contaminated solution; using a D-hank's balanced salt solution to clean cells; carrying out contamination on the cells, and culturing the contaminated cells in the incubator for 4 hours; c, adding a mixed solution of a DCFH-DA fluorescent probe and the culture medium, sealing and wrapping with tin foil paper, and culturing the mixed solution in the incubator for 0.5 hour; d, observing the fluorescence intensity and the fluorescence distribution under an inverted fluorescence microscope, and photographing; and e, processing images by fluorescence analysis software to obtain initial data, analyzing and comparing. According to the method, the cell section preparation is fast and simple, and the raw material is easy to buy; by the cell detection method, the result is accurate, the influence factor is fewer, and multiple nano particles can be detected simultaneously; and laboratory reagents are safe and reliable and have no strong toxicity.
Description
Technical field
The present invention relates to the method for a kind of accurate detection atmosphere nano particle and industrial nano granular cell toxicity difference, specifically belong to Atmospheric particulates and industrial nano particle contamination risk assessment technical field.
Background technology
Atmospheric particulates have become the primary pollutant of China's urban atmosphere, and atmosphere nano particle (or atmosphere ultra-fine grain) is the Atmospheric particulates important component parts.The industrial nano particle is meant the particle of the yardstick Chu Yu ≦ 100nm that in three dimensions, has one dimension at least; The industrial nano particle has been widely used in the fields such as coating, cosmetics, catalyzer, medical science at present; The industrial nano particle can have a mind to or be dispersed into unintentionally in the atmosphere in the process of producing and using, and becomes the part of atmosphere nano particle.The research of atmosphere nano particle and industrial nano particle health effect has been become the research focus of ecotoxicology, environmental science etc.(3-(4 for traditional research method such as MTT; 5)-dimethylthiahiazo is (z-yl)-3; 5-di-phenytetrazoliumromide, tetrazolium bromide), there are some shortcomings in LDH evaluation methods such as (lactate dehydrogenase, lactic dehydrogenases); Not directly perceived like result's performance; Influence factor is more, and accuracy is not high, needs repeatedly repeated experiments; Consuming time more, there is carcinogenicity etc. in experiment reagent.Therefore need seek new ways and means detects and estimates the cytotoxicity of atmosphere nano particle and industrial nano particle.
Summary of the invention
To defective of the prior art, the purpose of this invention is to provide a kind of method that detects atmosphere nano particle and industrial nano granular cell toxicity difference.This cell experiment detection method is directly simple, can detect multiple nano particle simultaneously; Its result is shown as image and data, makes the cytotoxicity difference visual pattern of atmosphere nano particle and industrial nano particle, and accuracy is high.
Basic design of the present invention is:
Utilize DCFH-DA (2`; 7`-dichlorodihydrofluorescin diacetate; 2`, 7`-dichlorofluorescein diacetate) to detect the content of intracellular reactive oxygen be the Perfected process of distinguishing atmosphere nano particle and industrial nano granular cell toxicity difference to method.(reactive oxygen species ROS) generally comprises hydrogen oxide, superoxide radical, hydroxy radical etc. to active oxygen, and the too high meeting of active oxygen causes cytotoxicity, causes permeability of cell membrane change, dna damage etc.DCFH-DA is a kind of chemical substance of nonpolar, non-fluorescence; Can permeates cell membranes; By intracellular enzymatic degradation is reductibility dichlorofluorescein (2`; 7`-dichlorodihydrofluorescin; DCFH); DCFH can by active oxygen (ROS) free-radical oxidation become high-efficiency fluorescence material oxidisability dichlorofluorescein (2`, 7`-dichlorofluorescin, DCF).Can learn the content of intracellular reactive oxygen through fluorescence intensity, therefore, the Cytotoxic difference of utilizing the DCFH-DA method to distinguish atmosphere nano particle and industrial nano particle has become possibility.
In order to achieve the above object, based on above inventive concept, the present invention adopts following technical proposals:
A kind of method that detects atmosphere nano particle and industrial nano granular cell toxicity difference, its sequence of operation and step are following:
A. the cell that gets off with trypsinization is sucked in the centrifuge tube, abandoning supernatant adds nutrient culture media and is made into cell suspending liquid, and with liquid-transfering gun piping and druming cell is uniformly dispersed, and is inoculated in the Tissue Culture Dish, places incubator to cultivate 24 hours double dish;
B. the particle solution with preparation mixes with nutrient culture media, is made into contamination solution; Use liquid-transfering gun with waste liquid sucking-off in the Tissue Culture Dish, clean cell with the D-hank`s balanced salt solution; Draw the contamination drips of solution with liquid-transfering gun then and be added in the Tissue Culture Dish pair cell and contaminate, put into incubator and cultivated 4 hours;
C. clean cell with the D-Hank`s balanced salt solution, add the mixed solution of DCFH-DA fluorescence probe and nutrient culture media then respectively,, put into incubator and cultivated 0.5 hour with the airtight parcel of masking foil;
D. clean cell with the D-Hank`s balanced salt solution, under inverted fluorescence microscope, observe fluorescence intensity and fluorescence distribution, and take pictures;
E. obtaining primary data through fluorescence analysis software processes picture also analyzes relatively.
The present invention has following conspicuous outstanding substantive distinguishing features compared with prior art:
(1) the cell film-making is simple fast, and starting material are bought easily;
(2) the cell detection method result is accurate, and influence factor is less, and can detect multiple nano particle simultaneously;
(3) experiment reagent is safe and reliable, does not have bigger toxicity;
(4) result is shown as image and data, both can directly observe the power of fluorescence intensity come the Cytotoxic difference of comparative analysis through picture, also can come the difference of analysis of cells toxicity through data.
Description of drawings
Fig. 1 is for detecting the experimental principle figure of atmosphere nano particle and industrial nano granular cell toxicity difference;
Fig. 2 detects the size of atmosphere nanoparticles contamination component fluorescence intensity for embodiment 1 utilizes the DCFH-DA method;
Fig. 3 detects the size of industrial nano NiO particle contamination component fluorescence intensity for embodiment 1 utilizes the DCFH-DA method;
Fig. 4 detects the size of industrial nano ZnO particle contamination component fluorescence intensity for embodiment 1 utilizes the DCFH-DA method;
Fig. 5 detects industrial nano CeO for embodiment 1 utilizes the DCFH-DA method
2The size of particle contamination component fluorescence intensity;
Fig. 6 is the concrete fluorescence intensity data that draws after embodiment 1 handles through IPWIN60 fluorescence analysis software and Excel;
Fig. 7 detects the size of industrial nano NiO particle contamination component fluorescence intensity for embodiment 2 utilizes the DCFH-DA method;
Fig. 8 detects industrial nano CeO for embodiment 2 utilizes the DCFH-DA method
2The size of particle contamination component fluorescence intensity;
Fig. 9 is the concrete fluorescence intensity data that draws after embodiment 2 handles through IPWIN60 fluorescence analysis software and Excel.
Embodiment
Below in conjunction with description of drawings embodiments of the invention are described in further detail.
Embodiment 1
As shown in Figure 1, a kind of method that detects atmosphere nano particle and industrial nano granular cell toxicity difference, its sequence of operation and step are following:
A. the cell that gets off with trypsinization is sucked in the centrifuge tube, abandoning supernatant adds the nutrient culture media that contains 10% hyclone and is made into 1 * 10 of 3mL
5Individual/the mL cell suspending liquid, and cell is uniformly dispersed with liquid-transfering gun piping and druming, be inoculated in the Tissue Culture Dish, place incubator to cultivate 24 hours double dish;
B. with the industrial nano NiO, ZnO, the CeO that prepare
2Particle and atmosphere nanoparticles solution mix with nutrient culture media respectively, are made into the contamination solution of 4 kind of 50 μ g/mL; Use liquid-transfering gun with waste liquid sucking-off in the Tissue Culture Dish, clean cell with 2mL D-hank`s balanced salt solution; Draw the contamination drips of solution with liquid-transfering gun then and be added in the Tissue Culture Dish pair cell and contaminate, put into incubator and cultivated 4 hours;
C. clean cell with 2mL D-Hank`s balanced salt solution; The mixed solution (volume ratio of fluorescence probe and nutrient culture media is 1:2100) that adds 2mL DCFH-DA fluorescence probe and nutrient culture media is then respectively put into incubator with the airtight parcel of masking foil and was cultivated 0.5 hour;
D. utilize the D-Hank`s balanced salt solution to clean cell, under inverted fluorescence microscope, observe fluorescence intensity and fluorescence distribution, take pictures;
E. handle picture through fluorescence analysis software I PWIN60 and obtain primary data, use Excel to analyze relatively.
Like Fig. 2, Fig. 3, Fig. 4, shown in Figure 5; Be the instance 1 described picture of being clapped behind the inverted fluorescence microscope observation cell that utilizes; Fig. 6 obtains primary data for using IPWIN60 to handle picture; With the data plot after the EXCEL comparative analysis; The result shows that the fluorescence intensity size is followed successively by in the particle contamination component cells: Na MiNiO>Na MiZnO>Atmosphere Na Mikeli>Nano Ce O
2, promptly the size of particle pair cell toxicity is: Na MiNiO>Na MiZnO>Atmosphere Na Mikeli>Nano Ce O
2
Embodiment 2
In this example, cellular incubation is consistent with embodiment 1 with inoculation, and the contamination process is slightly different, and difference is to be made into the contamination solution of 100 μ g/mL, and the contamination component has only industrial nano NiO and nano Ce O
2Particle.
Like Fig. 7, shown in Figure 8; Be the instance 2 described pictures of being clapped behind the inverted fluorescence microscope observation cell that utilize, Fig. 9 obtains primary data for using IPWIN60 to handle picture, and the result shows; Industrial nano NiO particle contamination component fluorescence intensity is stronger, industrial nano CeO
2Particle contamination component fluorescence intensity a little less than, but along with the increase of concentration of contamination, industrial nano NiO particle and CeO
2Fluorescence intensity than strengthening to some extent among the embodiment 1.
Claims (1)
1. method that detects atmosphere nano particle and industrial nano granular cell toxicity difference is characterized in that its sequence of operation and step are following:
A. the cell that gets off with trypsinization is sucked in the centrifuge tube, abandoning supernatant adds nutrient culture media and is made into cell suspending liquid, and with liquid-transfering gun piping and druming cell is uniformly dispersed, and is inoculated in the Tissue Culture Dish, places incubator to cultivate 24 hours double dish;
B. the particle solution with preparation mixes with nutrient culture media, is made into contamination solution; Use liquid-transfering gun with waste liquid sucking-off in the Tissue Culture Dish, clean cell with the D-hank`s balanced salt solution; Draw the contamination drips of solution with liquid-transfering gun then and be added in the Tissue Culture Dish, put into incubator and cultivated 4 hours;
C. clean cell with the D-Hank`s balanced salt solution, add the mixed solution of DCFH-DA fluorescence probe and nutrient culture media then respectively,, put into incubator and cultivated 0.5 hour with the airtight parcel of masking foil;
D. clean cell with the D-Hank`s balanced salt solution, under inverted fluorescence microscope, observe fluorescence intensity and fluorescence distribution, and take pictures;
E. obtain primary data and comparative analysis through fluorescence analysis software processes picture.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104569068A (en) * | 2014-12-26 | 2015-04-29 | 杭州师范大学 | Evaluation method for fog and haze pollutant toxicity |
| CN105548114A (en) * | 2015-12-31 | 2016-05-04 | 北京大学 | Method for analyzing atmospheric particulate matter toxicity online in real time |
| CN106290272A (en) * | 2016-07-25 | 2017-01-04 | 北京大学 | A kind of method of real-time detection Atmospheric particulates bio-toxicity |
| CN114292753A (en) * | 2021-12-23 | 2022-04-08 | 南京大学 | Method for separating cells and discharging nanoparticles from cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101671718A (en) * | 2009-10-27 | 2010-03-17 | 北京师范大学 | Detection method of toxicity of nanomaterials |
| CN102012380A (en) * | 2010-09-21 | 2011-04-13 | 上海大学 | Method for detecting distribution of poisonous heavy metal elements in cells |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101671718A (en) * | 2009-10-27 | 2010-03-17 | 北京师范大学 | Detection method of toxicity of nanomaterials |
| CN102012380A (en) * | 2010-09-21 | 2011-04-13 | 上海大学 | Method for detecting distribution of poisonous heavy metal elements in cells |
Non-Patent Citations (1)
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| 吴秋云等: "不同粒径纳米二氧化硅的体外细胞膜毒性作用", 《中国生物医学工程学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104569068A (en) * | 2014-12-26 | 2015-04-29 | 杭州师范大学 | Evaluation method for fog and haze pollutant toxicity |
| CN104569068B (en) * | 2014-12-26 | 2017-09-26 | 杭州师范大学 | A kind of haze pollutant toxicity assessment method |
| CN105548114A (en) * | 2015-12-31 | 2016-05-04 | 北京大学 | Method for analyzing atmospheric particulate matter toxicity online in real time |
| CN105548114B (en) * | 2015-12-31 | 2018-07-13 | 北京大学 | A method of based on saccharomycete on-line analysis Atmospheric particulates toxicity |
| CN106290272A (en) * | 2016-07-25 | 2017-01-04 | 北京大学 | A kind of method of real-time detection Atmospheric particulates bio-toxicity |
| CN114292753A (en) * | 2021-12-23 | 2022-04-08 | 南京大学 | Method for separating cells and discharging nanoparticles from cells |
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Application publication date: 20120208 |