CN108220382A - It is a kind of using palladium nano-complex evaluation of flue gas exposure to the method for cellular damage - Google Patents
It is a kind of using palladium nano-complex evaluation of flue gas exposure to the method for cellular damage Download PDFInfo
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- CN108220382A CN108220382A CN201810003812.XA CN201810003812A CN108220382A CN 108220382 A CN108220382 A CN 108220382A CN 201810003812 A CN201810003812 A CN 201810003812A CN 108220382 A CN108220382 A CN 108220382A
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- flue gas
- palladium nano
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
Abstract
The method of cellular damage is included the following steps the invention discloses palladium nano-complex evaluation of flue gas exposure is used：A, Pd nano particle is well-dispersed in hyaluronic acid colloid aqueous solution, cryospray drying then is carried out to mixture, the surface disperseed is coated with the Pd nano particle of hyaluronic acid gel, as described palladium nano-complex；B, make palladium nano-complex described in cellular uptake to be measured；The palladium nano-complex is combined with being catalyzed hair clip assemble method with mRNA simultaneously and then by base pair complementarity principle progress gene recombination formation mRNA DNA after intake, can be fluoresced；Then, C, above-mentioned palladium nano-complex is added in into the sample solution comprising subject cell and is stood enough for a long time, then before and after flue gas trapping object is exposed to, the fluorescence intensity that cell is sent out is measured respectively, according to the two ratio-dependent apoptosis rate, and then evaluation of flue gas exposure is to the degree of injury of cell.
Description
Technical field
The invention belongs to biological detecting method fields.
Background technology
During the smog that people's drawing tobacco product is sent out, respiratory tract cell can be when being directly exposed to flue gas by different journeys
The damage of degree.Tobacco enterprise has been working hard carry out harm reduction processing to tobacco to reduce this damage.It has been developed for
Various harm reduction methods in order to evaluate the quality of harm reduction effect, need a kind of method of evaluation of flue gas exposure to cellular damage,
Preferably quantitative method.
Conventional method is that human respiratory tract's cell (such as people's Thorium Lung Burden system A549 cells) is taken to be allowed to exposed
In flue gas, by detecting the quantity of apoptotic cell after fume exposure, carry out degree of injury of the evaluation of flue gas exposure to cell.This method
In need to dye cell DNA using fluorescent dye, and then analyze active somatic cell quantity.But fluorescent dye
The activity of cell can be destroyed and lead to the generation of intracellular toxin, this may carry out the result that electronic cigarette smog cell exposes
Interference affects the accuracy of evaluation result.
For this reason, it may be necessary to propose method of the better evaluation of flue gas exposure to cellular damage.
Invention content
The present invention provides it is a kind of using palladium nano-complex evaluation of flue gas exposure to the method for cellular damage, including as follows
Step：
A, Pd nano particle is well-dispersed in hyaluronic acid colloid aqueous solution, cryospray then is carried out to mixture
Dry, the surface disperseed is coated with the Pd nano particle of hyaluronic acid gel, as described palladium nano-complex；
B, make palladium nano-complex described in cellular uptake to be measured；The palladium nano-complex is to be catalyzed hair clip assembling side after intake
Method is combined with mRNA, and the mRNA for being combined with the palladium nano-complex is formed by base pair complementarity principle progress gene recombination
MRNA-DNA forms the cell that can be fluoresced under extraneous thermal effect；
C, above-mentioned palladium nano-complex is added in into the sample solution comprising subject cell and is stood enough for a long time, then
Before and after flue gas trapping object is exposed to, the fluorescence intensity that cell is sent out is measured under light radiation respectively, according to the two ratio
Apoptosis rate before and after the determining fume exposure of example, and then evaluation of flue gas exposure is to the degree of injury of cell.
Each step is specific as follows：
A, the preparation of composite Nano palladium
The present invention selects Pd nano particle to be combined the palladium nanometer for preparing lossless cell as carrier and high molecular material
Particle composite, nanoparticle palladium have typical nano material characteristic, i.e., larger specific surface area, surface is covered with stepped knot
Structure, this structure have the unstability atom of high surface energy, i.e., there are many unsatisfied chemical bonds, this provides more adsorption potential
Point and action site, easily pass through chemical bonds with foreign molecules.Pd nano particle has the category of fluorescent media in itself simultaneously
Property can provide fluorescence in genetic test, avoid the cytotoxicity brought using chemiluminescence agent, can be realized using the present invention
The non-destructive testing of cell target gene.
Using in-situ synthesis, by three-dimensional composite stirring technology by hyaluronic acid gel macromolecule network system with receiving
The rice full and uniform mixing of palladium particle, is prepared into palladium nanoparticles hyaluronic acid, and form stable complex.
Specific synthesis step is as follows：Under light at room temperature catalytic condition, using nanoparticle palladium and hyaluronic acid gel into
Row three-dimensional hybrid stirs, and 90 revs/min of speed is mixed.Use scanning electron microscope characterization hyaluronic acid gel and palladium nanometer
The combination degree of grain.Treat that nanoparticle palladium reaches homodisperse in hyaluronic acid gel and can stop stirring.It obtains uniformly
Freeze-drying process is carried out to it after stable Technique of Nano Pd/hyaluronic acid gel compound, finally obtains Technique of Nano Pd/hyalomitome
Sour composite particles, are shown in Fig. 1.
B, Technique of Nano Pd/hyaluronic acid assembling
By the palladium nanometer transparent matter acid composite particles prepared, using the assembling of catalysis hair clip, (CHA, one kind is without enzymatic
Amplification method) method combined with DNA preparation become nanoanalysis platform.Technique of Nano Pd hyaluronic acid in specific implementation process
Grain is combined using catalysis hair clip assemble method with mRNA.MRNA with palladium nano-particles hyaluronic acid is mutual by base
It mends pair principle and carries out gene recombination formation mRNA-DNA.
The effect of palladium nanometer transparent matter acid composite particles is illumination medium.The nano platform can not only increase sensitivity
It can also make mRNA specific imagings in cell.The palladium nanometer transparent matter acid composite particles of the present invention and the area of mRNA survivins
It is not that survivin has different efficiency in different cells, and palladium nanometer transparent matter acid composite particles provide height by radiation energy
The illumination medium of effect.The use of the palladium nanometer transparent matter acid composite particles is high sensitivity and specific good intracellular imaging
Medium.
C, fluorescence intensity detects
Above-mentioned palladium nano-complex is added in into the sample solution comprising subject cell and is stood enough for a long time, Ran Hou
It is exposed to before and after flue gas trapping object, the fluorescence intensity that cell is sent out is measured under light radiation respectively, according to the two ratio
Determine the apoptosis rate before and after fume exposure, and then evaluation of flue gas exposure is to the degree of injury of cell.
Beneficial effects of the present invention：
1st, the present invention wraps up Pd nano particle using hyaluronic acid gel so that it will not be killed by after cellular uptake
Cell, but can be stable in the presence of in active somatic cell.If the hyaluronic acid gel does not wrap up, Pd nano particle can be because
Its strong bactericidal properties and kill cell to be measured.
2nd, the use of cytotoxic conventional fluorescent coloring agent is avoided so that measurement result is more accurate.
Description of the drawings
Fig. 1 is that (a, b, c, d are different between compound for the stereoscan photograph of palladium nano-complex that is prepared in the present invention
Bonding state).
Specific embodiment
Embodiment 1
First, it is prepared as follows the Pd nano particle that surface is coated with hyaluronic acid.
The use of length is about the spherical Technique of Nano Pd that 50-70nm diameters are about 40-60nm under the conditions of room temperature and photocatalysis
Grain is mixed with hyaluronic acid gel, and 90 revs/min of speed is mixed.Hyaluronic acid is characterized using scanning electron microscope
The combination degree of hydrogel and palladium nano-particles.Treat that nanoparticle palladium reaches homodisperse in hyaluronic acid gel and can stop
Only stir.Freeze-drying process is carried out to it after obtaining uniform and stable Technique of Nano Pd/hyaluronic acid gel compound, it is final to obtain
To Technique of Nano Pd/hyaluronic acid particle, see that (a, b, c, d are respectively that the Pd nano particle of different connection forms is compound to Fig. 1
Object).Then, using Coresta suction modes, the flue gas generated after being heated with cambridge filter trapping novel tobacco product, then
The flue gas trapped is transferred in solution by soaked in solvent cambridge filter, as solution to be measured.Take people's alveolar type epithelium thin
Born of the same parents system A549 cells are as subject cell.
Culture solution containing above-mentioned cell is divided into two parts, portion is directly added into Technique of Nano Pd/hyaluronic acid of the present invention
Composite particles, Technique of Nano Pd of the invention/hyaluronic acid particle can be combined with the mRNA in active somatic cell, Jin Erjie
The mRNA that closing has Technique of Nano Pd/hyaluronic acid particle carries out gene recombination by base pair complementarity principle and forms mRNA-
DNA, the mRNA-DNA for being combined with Technique of Nano Pd/hyaluronic acid particle can be sent out in the case where argon ion laser sends out laser irradiation
Go out the generation transition of metallic outer-shell electron and send out fluorescence, fully after reaction, measure its initial fluorescent intensity.
Another then adds in the above-mentioned solution to be measured containing flue gas trapping object into cell culture fluid, and is deposited in suitable cell
After enough long-times are stood at temperature living, Technique of Nano Pd/hyaluronic acid particle of the present invention is added, due to the present invention
Technique of Nano Pd/hyaluronic acid particle can be combined with the mRNA in active somatic cell, and then be combined with Technique of Nano Pd/hyalomitome
The mRNA of sour composite particles carries out gene recombination by base pair complementarity principle and forms mRNA-DNA, and the cell die is then
Above-mentioned cohesive process will not occur, therefore by fluorescence intensity and compared with initial fluorescent intensity, it will be able to it is convenient and efficient
The cell proportion of ground detection survival.With the ratio of this survivaling cell, carry out the cytotoxicity of evaluation of flue gas trapping object.
Comparative example 1
It is wrapped up using same Pd nano particle, but without hyaluronic acid, as a result, it has been found that, into subject cell, addition palladium is received
After rice corpuscles, due to the powerful bactericidal effect of Pd nano particle, subject cell is all killed, and can not carry out subsequent experimental.
Claims (2)
1. it is a kind of using palladium nano-complex evaluation of flue gas exposure to the method for cellular damage, which is characterized in that including walking as follows
Suddenly：
A, Pd nano particle is well-dispersed in hyaluronic acid colloid aqueous solution, then carrying out cryospray to mixture does
Dry, the surface disperseed is coated with the Pd nano particle of hyaluronic acid gel, as described palladium nano-complex；
B, make palladium nano-complex described in cellular uptake to be measured；After intake the palladium nano-complex be catalyzed hair clip assemble method with
MRNA is combined, and the mRNA for being combined with the palladium nano-complex carries out gene recombination formation mRNA- by base pair complementarity principle
DNA forms the cell that can be fluoresced under extraneous thermal effect；Then
C, above-mentioned palladium nano-complex is added in into the sample solution comprising subject cell and is stood enough for a long time, then sudden and violent
It is exposed to before and after flue gas trapping object, measures the fluorescence intensity that cell is sent out under light radiation respectively, it is true according to the two ratio
Determine the apoptosis rate before and after fume exposure, and then evaluation of flue gas exposure is to the degree of injury of cell.
2. according to the method described in claim 1, it is characterized in that, the Pd nano particle be rod-like nano particle, length
For 50-70nm, a diameter of 40-60nmm.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810003812.XA CN108220382B (en) | 2018-01-03 | 2018-01-03 | Method for evaluating damage of smoke exposure to cells by using palladium nano composite |
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| CN201810003812.XA CN108220382B (en) | 2018-01-03 | 2018-01-03 | Method for evaluating damage of smoke exposure to cells by using palladium nano composite |
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| CN108220382B CN108220382B (en) | 2020-07-21 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104931473A (en) * | 2015-06-16 | 2015-09-23 | 云南中烟工业有限责任公司 | Evaluation method for measuring cell DNA damage caused by soluble heavy metal |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104931473A (en) * | 2015-06-16 | 2015-09-23 | 云南中烟工业有限责任公司 | Evaluation method for measuring cell DNA damage caused by soluble heavy metal |
Non-Patent Citations (4)
| Title |
|---|
| FENG-XIA SU ET AL.: "Intracellular Messenger RNA Triggered Catalytic Hairpin Assembly for Fluorescence Imaging Guided Photothermal Therapy", 《ANAL. CHEM.》 * |
| LINDSAY RAMAGE ET AL.: "Induction of apoptosis with tobacco smoke and related products in A549 lung epithelial cells in vitro", 《JOURNAL OF INFLAMMATION》 * |
| 李棒棒: "用于肿瘤诊断治疗的透明质酸基多功能纳米粒子的研究", 《万方学位论文数据库》 * |
| 龚守良: "《电离辐射生殖遗传效应》", 31 October 2009, 原子能出版社 * |
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