CN106834429A - For confirming less than the 2.5 microns whether exposed biomarkers of fine dust and utilizing its confirmation method - Google Patents
For confirming less than the 2.5 microns whether exposed biomarkers of fine dust and utilizing its confirmation method Download PDFInfo
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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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The present invention relates to a kind of gene expression because of 2.5 microns (μm) following fine dust (particulate matter 2.5,PM2.5 the biomarker for) and specifically increasing or decreasing and the specific method for whether exposing using fine dust below 2.5 microns of its confirmation,Specifically,Lung tissue test portion is gathered from the mouse model exposed to two kinds of different PM2.5 of actual characteristic (water-soluble or Organic),Mouse 55 is integrated with to utilize,More than 681 less than the 2.5 microns gene expression profiles of fine dust of the oligosaccharides microarray of gene,Analyze the changes in gene expression of the test portion,Results verification exposed to PM2.5 because expressing change jointly 25 kinds of genes,Such that it is able to the gene is used as biomarker,It is usefully used for monitoring and judging the PM2.5 pollutions in air,Can serve as the instrument of the toxic effect mechanism that prediction induces by PM2.5.
Description
Technical field
The present invention relates to a kind of gene expression because of 2.5 microns (μm) following fine dust (particulate
Matter2.5, PM2.5) and the biomarker that specifically increases or decreases and using trickle ash below 2.5 microns of its confirmation
The method whether specificity of dirt exposes.
Background technology
Granular size for 10 μm (=0.001mm) dust below calls fine dust, major part is because of fuel combustion
It is super less than 2.5 that the fine dust (particulate matter) for artificially occurring is divided into PM10 of the diameter less than 10 and diameter
Fine dust PM2.5.In general, the occurring source major part of the PM2.5 that can be had a strong impact on to human body generation pole is because of fuel combustion
And occur, the ejected matter of boiler, automobile and power generating equipment etc. is main occurring source.Additionally, dispersed in building site, road etc.
Dust also accounts for many amounts.
PM2.5 sizes are smaller, and the translational speed in air is faster, and presumption about 1/3 at present is mobile over long distances, and 1/3 is this
Be expelled directly out, 1/3 results from the reaction in air.PM2.5 has feature of the diameter less than 2.5, thus visually is difficult to differentiate,
Feature with deep intrusion alveolar.PM2.5 mainly enters human body by breathing, occurs because of fuel combustion, therefore can be in work
Road, power generating equipment that factory or traffic mix etc. over-exposure.
In addition, PM2.5 is to cause one of material of tuberculosis by respiratory system, when long-time exposes, particle is thinner
It is micro-, do not filtered by schneiderian membrane, upon inhalation, directly invade to alveolar, the original of the diseases such as asthma, idiocrasy, lung cancer can be turned into
Cause, can increase Infant Mortality and produce influence.Due to this harmfulness, in the IAQ administrative law of communal facility etc.
In, for the pleasant room air of newly-built apartment and flats, the atmospheric environment benchmark of PM2.5 be set to average annual below 25g/m3,
Average daily below 50g/m3.Now, PM2.5 increases the report that the influence that health is produced further increases, therefore, Ministry of environment
Report claims, and new environmental criteria will be introduced from the current year.
In on domestic air for the data of the dustiness of PM2.5, because mobility is big in atmosphere, each area
The difference such as distributed density, composition it is big, due in superfine dusty material characteristic, to this survey data very not
Foot.Therefore, Ministry of environment be devoted in order to formulate effective countermeasure, collect PM2.5 long range movement present situation, each department into
Divide analysis of data, largely to discharge unit as preferential objects carry out real random variables and game.
In addition, the analysis of human toxicity degree, constituent and changes in gene expression to sandstorm, fine dust PM10 etc.
Although research some report, constituent analysis is almost had been limited to the research of superfine dust PM2.5.
Although as described above, potential hazards of the PM2.5 to the mankind, the Evaluation of Harmfulness data of PM2.5 are insufficient, when
It is assumed that when the PM2.5 concentration levels of WHO suggestions reach air quality benchmark, calculating and being expected to what is realized because improving PM2.5 concentration
Reducing the analysis method of the health benefits of Deaths has not certainty and limitation.
The plan of several genes group (genome) base sequence such as 6 kinds of mammality, 292 kinds of microorganism is completed and has reported NCBI
(National Center for Biotechnology Information, American National Biotechnology Information center), in order to
Based on the data of such flood tide for obtaining, the function of gene is studied, carried out full-length genome expression (genome-wide
Expression) study.In order to analyze the expression of thousands of genes by once testing, DNA microarray is performed
(microarray) (Proc.Natl.Acad.Sci.USA 93 is analyzed：10614-10619,1996).
Microarray (microarray) is cDNA (complementary DNA, complementary DNA (cDNA)) or 20-25
The fragment of the oligonucleotide (oligonucleotide) of base-pair (base pair) length is integrated in glass.CDNA microarrays
It is, by the company such as the research department in school or Agilent, Genomic Solutions, mechanically cDNA gleanings to be fixed
In on chip, or (J.Am.Acad.Dermatol.51 is produced using inkjet printing (ink jetting) method：681-692,
2004).Oligonucleotide microarray is that Affymetrix company utilizes photoetching process (photolithography), by means of on chip
The method that is directly synthesized and manufacture, in Agilent company etc., carried out with to the method that the oligonucleotide for synthesizing is fixed
Production (J.Am.Acad.Dermatol.51：681-692,2004).
In order to analyze the expression of gene, RNA is obtained from test portions such as tissue, cells, with the oligonucleotide in DNA microarray
Perform hybridization reaction.The RNA of acquisition is marked using fluorescence or isotope, is converted into cDNA.Oligosaccharides microarray is main with two
Plant different fluorescence (examples：Cy3 (cyanine 3) and Cy5) respectively mark control group and experimental group, on the same chip simultaneously
After performing hybridization reaction, optical scanner image obtains the intensity of fluorescence, and its result is analyzed.According to two kinds of fluorescence intensities
Ratio, determine gene expression whether (Genomics Proteomics I：1-10,2002).
Recently, with the toxicogenomics (Toxicogenomics) as the sophisticated skill and technique using DNA microarray technology
Research etc. integrates with, and is not only the pharmaceuticals and new drug candidates matter of a large amount of outputs (high throughput), and including representing
Property environmental pollutants including, can carry out because caused by all chemical substances in particular organization or cell line express gene
Expression pattern analysis, quantitative analysis.Therefore, in specific cells, the expression frequency of specific gene is analyzed such that it is able to excavate
The gene related to the illeffects of side effects of pharmaceutical drugs and environmental pollutants, thereby, it is possible to understand environmental pollutants
Illeffects and the molecular mechanism of medicine effect and side effect, and then can retrieve and confirm to induce the thing of toxicity and side effect
Matter.
Therefore, the present inventor is striven to find during being confirmed whether exposed method for less than 2.5 microns fine dusts,
Lung tissue test portion is acquired from the mouse model exposed to two kinds of different PM2.5 of actual characteristic (water-soluble or Organic), with
Using mouse 55 is integrated with, more than 681 less than the 2.5 microns gene expression profiles of fine dust of the oligosaccharides microarray of gene divide
Analyse the changes in gene expression of the test portion, results verification because expressing change jointly 25 kinds of genes exposed to PM2.5,
So as to find the genome can be used as to the biomarker exposed to PM2.5 and the side for utilizing its confirmation whether to expose
Method, this completes the present invention.
The content of the invention
(technical problem to be solved)
It is an object of the invention to provide it is a kind of because less than 2.5 microns fine dusts (particulate matter2.5,
PM2.5 exposure) and specifically the biomarker of overexpression or low expression and confirm that PM2.5 is special using the biomarker
The method whether property exposes.
(means of solve problem)
To reach the purpose, the present invention provides a kind of gene being integrated with more than a certain kind selected from following groups or its is mutual
Whether 2.5 microns (μm) following fine dust (particulate matter 2.5, PM2.5) of benefit chain molecule exposes and confirms to use
Micro-array chip (microarray chip)：
Gene accession number (Genebank) NM_001163145 (1810041L15Rik, RIKEN cDNA
1810041L15gene), gene accession number (Genebank) NM_001081381 (2610002I17Rik, RIKEN cDNA
2610002I17), gene accession number (Genebank) NM_176952 (6430573F11Rik, RIKEN cDNA
6430573F11gene), gene accession number (Genebank) AK141429 (BC023202, cDNA sequence
BC023202), gene accession number (Genebank) NM_144890 (Bpifb5, BPI fold containing family B,
Member 5), gene accession number (Genebank) NM_178626 (Cdc42se2, CDC42small effector 2), gene
Registration number (Genebank) NM_199032 (Cep135, centrosomal protein135), gene accession number (Genebank)
NM_146023 (Evi2b, ecotropic viral integration site 2b), gene accession number (Genebank)
AK146791 (Ewsr1, Ewing sarcoma breakpoint region 1), gene accession number (Genebank) NM_
197989 (Fam58b, family with sequence similarity 58, member B), gene accession number
(Genebank) NM_010200 (Fgf13, fibroblast growth factor 13), gene accession number (Genebank)
NR_003248 (Foxl2os, forkhead box L2opposite strand transcript), gene accession number
(Genebank) NR_037689 (Gm1968, predicted gene 1968), gene accession number (Genebank) XM_
001472451 (Gm2102, predicted gene 2102), gene accession number (Genebank) AK078977 (Mapkapk3,
Mitogen-activated protein kinase-activated protein kinase 3), gene accession number
(Genebank) NM_001037906 (Nell1, NEL-like 1 (chicken)), gene accession number (Genebank) NM_
001134480(Plcxd2,phosphatidylinositol-specific phospholipase C,X domain
Containing 2), gene accession number (Genebank) NM_008895 (Pomc, pro-opiomelanocortin-alpha),
Gene accession number (Genebank) NM_011198 (Ptgs2, prostaglandin-endoperoxide synthase 2), base
Because of registration number (Genebank) NM_028762 (Rbm19, RNA binding motif protein19), gene accession number
(Genebank) NM_033354 (Sec16b, SEC16homolog B (S.cerevisiae)), gene accession number (Genebank)
NM_001254731 (Sgsm1, small G protein signaling modulator 1), gene accession number
(Genebank) NM_199199 (Tmem199, transmembrane protein 199), gene accession number (Genebank)
NM_026454 (Ube2f, ubiquitin-conjugating enzyme E2F (putative)) makees gene accession number
(Genebank)NM_026615(Urm1,ubiquitin related modifier 1homolog(S.cerevisiae))。
In addition, the present invention provide the exposure of a kind of 2.5 microns (μm) comprising the micro-array chip following fine dust with
No confirmation kit.
In addition, the present invention provides a kind of comprising can be with following gene complementations, micro- to the 2.5 of the primer of following gene magnifications
Whether the following fine dust of rice exposes and confirms to use kit：
Gene accession number (Genebank) NM_001163145 (1810041L15Rik, RIKEN cDNA
1810041L15gene), gene accession number (Genebank) NM_001081381 (2610002I17Rik, RIKEN cDNA
2610002I17), gene accession number (Genebank) NM_176952 (6430573F11Rik, RIKEN cDNA
6430573F11gene), gene accession number (Genebank) AK141429 (BC023202, cDNA sequence
BC023202), gene accession number (Genebank) NM_144890 (Bpifb5, BPI fold containing family B,
Member 5), gene accession number (Genebank) NM_178626 (Cdc42se2, CDC42small effector 2), gene
Registration number (Genebank) NM_199032 (Cep135, centrosomal protein135), gene accession number (Genebank)
NM_146023 (Evi2b, ecotropic viral integration site 2b), gene accession number (Genebank)
AK146791 (Ewsr1, Ewing sarcoma breakpoint region 1), gene accession number (Genebank) NM_
197989 (Fam58b, family with sequence similarity 58, member B), gene accession number
(Genebank) NM_010200 (Fgf13, fibroblast growth factor 13), gene accession number (Genebank)
NR_003248 (Foxl2os, forkhead box L2opposite strand transcript), gene accession number
(Genebank) NR_037689 (Gm1968, predicted gene 1968), gene accession number (Genebank) XM_
001472451 (Gm2102, predicted gene 2102), gene accession number (Genebank) AK078977 (Mapkapk3,
Mitogen-activated protein kinase-activated protein kinase 3), gene accession number
(Genebank) NM_001037906 (Nell1, NEL-like 1 (chicken)), gene accession number (Genebank) NM_
001134480(Plcxd2,phosphatidylinositol-specific phospholipase C,X domain
Containing 2), gene accession number (Genebank) NM_008895 (Pomc, pro-opiomelanocortin-alpha),
Gene accession number (Genebank) NM_011198 (Ptgs2, prostaglandin-endoperoxide synthase 2), base
Because of registration number (Genebank) NM_028762 (Rbm19, RNA binding motif protein19), gene accession number
(Genebank) NM_033354 (Sec16b, SEC16homolog B (S.cerevisiae)), gene accession number (Genebank)
NM_001254731 (Sgsm1, small G protein signaling modulator 1), gene accession number
(Genebank) NM_199199 (Tmem199, transmembrane protein 199), gene accession number (Genebank)
NM_026454 (Ube2f, ubiquitin-conjugating enzyme E2F (putative)) and gene accession number
(Genebank) NM_026615(Urm1,ubiquitin related modifier 1homolog(S.cerevisiae))。
Meanwhile, the present invention provides a kind of less than 2.5 microns fine dusts and whether exposes confirmation method, including：
1) thin from the body cell for as a corpse or other object for laboratory examination and chemical testing for experimental group separate and the body for from normal individual as a control group separate
The step of each RNA is separated in born of the same parents；
2) step 1) experimental group and the RNA of control group while synthesize cDNA, used each different glimmering
The step of stimulative substance is marked；
3) make step 2) with fluorescent material mark cDNA with DNA microarray chip hybridization of the invention the step of；
4) analytical procedure 3) reaction micro-array chip the step of；And
5) in step 4) analysis data in, the gene of the micro-array chip for being integrated in claim 1 of experimental group
Expression degree and control group the step of be compared confirmation.
(The effect of invention)
In the present invention, from exposed to two kinds of different (water-soluble or Organic) PM2.5 of respective characteristic
The mouse model collection lung tissue test portion of (particulate matter 2.5), is integrated with mouse 55, more than 681 base to utilize
Less than the 2.5 microns gene expression profiles of fine dust of the oligosaccharides microarray of cause, analyze the changes in gene expression of the test portion, knot
Fruit confirms, because expressing change jointly 25 kinds of genes exposed to PM2.5, the genome can be usefully used as
The method whether exposed to the biomarker exposed to PM2.5 and using its confirmation.
Brief description of the drawings
Fig. 1 is in 2.5 microns (μm) following fine dust (particulate matter 2.5, PM2.5) treatment
The figure in the abstraction process path of water-soluble extracting liquid and Organic extract solution in group.
Fig. 2 is to show complete in two kinds of mouse lung tissue test portions of PM2.5 using gene microarray chip analysis
The figure of the result of body gene expression pattern.
Fig. 3 is shown in using gene microarray chip analysis in two kinds of mouse lung tissue test portions of PM2.5
In the result of gene expression pattern, figure of the analytical table up to the result of the expression pattern of common 25 kinds of genes for changing more than 1.5 times.
Specific embodiment
The following detailed description of the present invention.
The present invention provide it is a kind of be integrated with it is micro- selected from gene more than following groups a certain or the 2.5 of its complementary strand molecule
Whether rice (μm) following fine dust (particulate matter 2.5, PM2.5) exposes and confirms to use micro-array chip
(microarray chip)：
Gene accession number (Genebank) NM_001163145 (1810041L15Rik, RIKEN cDNA
1810041L15gene), gene accession number (Genebank) NM_001081381 (2610002I17Rik, RIKEN cDNA
2610002I17), gene accession number (Genebank) NM_176952 (6430573F11Rik, RIKEN cDNA
6430573F11gene), gene accession number (Genebank) AK141429 (BC023202, cDNA sequence
BC023202), gene accession number (Genebank) NM_144890 (Bpifb5, BPI fold containing family B,
Member 5), gene accession number (Genebank) NM_178626 (Cdc42se2, CDC42small effector 2), gene
Registration number (Genebank) NM_199032 (Cep135, centrosomal protein135), gene accession number (Genebank)
NM_146023 (Evi2b, ecotropic viral integration site 2b), gene accession number (Genebank)
AK146791 (Ewsr1, Ewing sarcoma breakpoint region 1), gene accession number (Genebank) NM_
197989 (Fam58b, family with sequence similarity 58, member B), gene accession number
(Genebank) NM_010200 (Fgf13, fibroblast growth factor 13), gene accession number (Genebank)
NR_003248 (Foxl2os, forkhead box L2opposite strand transcript), gene accession number
(Genebank) NR_037689 (Gm1968, predicted gene 1968), gene accession number (Genebank) XM_
001472451 (Gm2102, predicted gene 2102), gene accession number (Genebank) AK078977 (Mapkapk3,
Mitogen-activated protein kinase-activated protein kinase 3), gene accession number
(Genebank) NM_001037906 (Nell1, NEL-like 1 (chicken)), gene accession number (Genebank) NM_
001134480(Plcxd2,phosphatidylinositol-specific phospholipase C,X domain
Containing 2), gene accession number (Genebank) NM_008895 (Pomc, pro-opiomelanocortin-alpha),
Gene accession number (Genebank) NM_011198 (Ptgs2, prostaglandin-endoperoxide synthase 2), base
Because of registration number (Genebank) NM_028762 (Rbm19, RNA binding motif protein19), gene accession number
(Genebank) NM_033354 (Sec16b, SEC16homolog B (S. cerevisiae)), gene accession number
(Genebank) NM_001254731 (Sgsm1, small G protein signaling modulator 1), gene registration
Number (Genebank) NM_199199 (Tmem199, transmembrane protein 199), gene accession number (Genebank)
NM_026454 (Ube2f, ubiquitin-conjugating enzyme E2F (putative)) and gene accession number
(Genebank)NM_026615(Urm1,ubiquitin related modifier 1homolog(S.cerevisiae))。
It is preferred that described 2.5 microns of (μm) following fine dusts are less than the 2.5 microns water-soluble or Organics of fine dust
Extract solution.
It is preferred that the fine dust is 2.5 microns of (μ for including superfine dust (ultra fine particle, UFP)
M) fine dust (particulate matter 2.5, PM2.5) below, most preferably 0.1 to 2.5 micron (μm) it is trickle
Dust, but it is not limited to this.
It is preferred that the fine dust extract solution is manufactured according to the manufacture method for comprising the steps, but it is not limited to this：
1) the step of trapping less than 2.5 microns fine dusts；
2) step 1) trapping fine dust addition Extraction solvent the step of extracted；And
3) to step 2) extract the step of filter.
In the process, preferred steps 2) Extraction solvent use water, alcohol, dichloromethane or their mixture.Make
It is the water, is preferably purified or distiller, most preferably with 3 times distilled water.As the alcohol, preferably by C1 to C2
Lower alcohol, as lower alcohol, preferably uses ethanol or methyl alcohol.As extracting method, preferably by ultrasonic grinder and stream high
Amount air sampler, but it is not limited to this.Additionally, it is preferred that the ultrasonic grind time is 20 to 60 minutes, more preferably 30 minutes,
But it is not limited to this.
Whether PM2.5 of the invention exposes confirmation micro-array chip can be with side known to those skilled in the art
Method makes.Method as the micro-array chip is made, in order to the biomarker explored is used as microprobe DNA molecular,
Fixed on the substrate of chip, preferably used ink-jet (inkjet) mode etc., more preferably used SurePrint inkjet
Micro dropping microarrays, but it is not limited to this.
It is preferred that the base plate coating of the DNA microarray chip has by epoxy resin (epoxy), amino silane (amino-
Silane a kind of), the free radical selected in the group that poly-l-lysine (poly-L-lysine) and aldehyde (aldehyde) are constituted,
But it is not limited to this.Additionally, it is preferred that the substrate is by slide, plastics, metal, silicones, nylon membrane and nitrocellulose membrane
Select to use in the group that (nitrocellulose membrane) is constituted, more preferably using the slide for being coated with epoxy resin,
But it is not limited to this.
In a particular embodiment of the present invention, it is (water-soluble or organic from two kinds of different PM2.5 of actual characteristic are exposed to
Property) mouse model collection lung tissue test portion, with using being integrated with mouse 55, more than 681 is the 2.5 of the oligosaccharides microarray of gene micro-
The gene expression profile of the following fine dust of rice, analyze the changes in gene expression of the test portion, and results verification is because being exposed to PM2.5
And express the 25 kinds of genes for changing jointly.
The genome therefore, it can usefully to be used as to the biomarker exposed to PM2.5.
In addition, the present invention provide a kind of exposure of 2.5 microns (μm) including the micro-array chip following fine dust with
No confirmation kit.
It is preferred that the kit is additional to include mouse pneumonocyte or lung tissue.
In addition, the present invention provides a kind of comprising can be with following gene complementations, micro- to the 2.5 of the primer of following gene magnifications
Whether the following fine dust of rice exposes and confirms to use kit：
Gene accession number (Genebank) NM_001163145 (1810041L15Rik, RIKEN cDNA
1810041L15gene), gene accession number (Genebank) NM_001081381 (2610002I17Rik, RIKEN cDNA
2610002I17), gene accession number (Genebank) NM_176952 (6430573F11Rik, RIKEN cDNA
6430573F11gene), gene accession number (Genebank) AK141429 (BC023202, cDNA sequence
BC023202), gene accession number (Genebank) NM_144890 (Bpifb5, BPI fold containing family B,
Member 5), gene accession number (Genebank) NM_178626 (Cdc42se2, CDC42small effector 2), gene
Registration number (Genebank) NM_199032 (Cep135, centrosomal protein135), gene accession number (Genebank)
NM_146023 (Evi2b, ecotropic viral integration site 2b), gene accession number (Genebank)
AK146791 (Ewsr1, Ewing sarcoma breakpoint region 1), gene accession number (Genebank) NM_
197989 (Fam58b, family with sequence similarity 58, member B), gene accession number
(Genebank) NM_010200 (Fgf13, fibroblast growth factor 13), gene accession number (Genebank)
NR_003248 (Foxl2os, forkhead box L2opposite strand transcript), gene accession number
(Genebank) NR_037689 (Gm1968, predicted gene 1968), gene accession number (Genebank) XM_
001472451 (Gm2102, predicted gene 2102), gene accession number (Genebank) AK078977 (Mapkapk3,
Mitogen-activated protein kinase-activated protein kinase 3), gene accession number
(Genebank) NM_001037906 (Nell1, NEL-like 1 (chicken)), gene accession number (Genebank) NM_
001134480(Plcxd2,phosphatidylinositol-specific phospholipase C,X domain
Containing 2), gene accession number (Genebank) NM_008895 (Pomc, pro-opiomelanocortin-alpha),
Gene accession number (Genebank) NM_011198 (Ptgs2, prostaglandin-endoperoxide synthase 2), base
Because of registration number (Genebank) NM_028762 (Rbm19, RNA binding motif protein19), gene accession number
(Genebank) NM_033354 (Sec16b, SEC16homolog B (S.cerevisiae)), gene accession number (Genebank)
NM_001254731 (Sgsm1, small G protein signaling modulator 1), gene accession number
(Genebank) NM_199199 (Tmem199, transmembrane protein 199), gene accession number (Genebank)
NM_026454 (Ube2f, ubiquitin-conjugating enzyme E2F (putative)) and gene accession number
(Genebank)NM_026615(Urm1,ubiquitin related modifier 1homolog(S.cerevisiae))。
Can be added in the kit comprising fluorescent material, preferably fluorescent material is selected from by Streptavidin-alkalescence
Phosphatase conjugate's matter (strepavidin-like phosphatease conjugate), chemiluminescence material
(chemiflurorensce) and the group that constitutes of chemiluminescent substance (chemiluminescent), but this is not limited to, at this
In the preferred embodiment of invention, Cy3, Cy5 have been used.
Can be added in the kit comprising reaction reagent, the reaction reagent can be by the buffering that is used in hybridization
Solution, for from the RNA synthesis reverse transcriptase of cDNA, cNTPs and rNTP (premixed type separates supply type), fluorescent dye
Mark reagent, washing buffer solution of chemical inducer etc. are constituted, but are not limited to this, and those skilled in the art is public
Including reaction reagent needed for the hybridization reaction of the DNA microarray chip known may each comprise.
In the present invention, it is thus identified that the gene of specific expressed change, thus its occur to less than 2.5 microns fine dusts
Can as be confirmed whether exposed to less than 2.5 microns fine dusts, less than 2.5 microns fine dust monitoring, less than 2.5 microns
Fine dust endangers sex determination and finds out because of the kit needed for the toxic effect mechanism that less than 2.5 microns fine dusts cause
And usefully use.
In addition, whether the present invention provides a kind of less than 2.5 microns fine dusts exposes confirmation method, including：
1) thin from the body cell for as a corpse or other object for laboratory examination and chemical testing for experimental group separate and the body for from normal individual as a control group separate
The step of each RNA is separated in born of the same parents；
2) step 1) experimental group and the RNA of control group while synthesize cDNA, used each different glimmering
The step of stimulative substance is marked；
3) make step 2) with fluorescent material mark cDNA with DNA microarray chip hybridization of the invention the step of；
4) analytical procedure 3) reaction micro-array chip the step of；And
5) in step 4) analysis data in, the gene of the micro-array chip for being integrated in claim 1 of experimental group
Expression degree and control group the step of be compared confirmation.
It is preferred that the step 1) body cell for mouse pneumonocyte or lung tissue, preferably described lung tissue be BALB/C it is small
Mouse lung tissue, preferably uses and receives to obtain person from each mouse group for being exposed to or being not exposed to PM2.5.
Additionally, it is preferred that the step 2) fluorescent material by Cy3 (cyanine 3), Cy5, the different sulphur cyanogen of poly-l-lysine
Sour fluorescein (poly L-lysine-fluorescein isothiocyanate, FITC), rhodamine B-isothiocyanate
Select to use in the group that (rhodamine-B-isothiocyanate, RITC) and rhodamine (rhodamine) are constituted, but do not limit
Due to this, fluorescent material known to those skilled in the art can be used.
Additionally, it is preferred that the step 4) analysis method using GeneSpring GX 12.6.1 softwares (Agilent, it is beautiful
State), but this is not limited to, even using analysis software known to those skilled in the art.
Additionally, it is preferred that the step 5) micro-array chip use mouse G3Mouse GE 8X 60K (Agilent, the U.S.)
Deng, but this is not limited to, as long as the gene that the expression described in the present invention in being equipped with murine genes changes jointly
The micro-array chip of (with reference to table 4), just can be used, the micro-array chip that most preferably with the present inventor makes.
Therefore, micro-array chip is make use of in the present invention, and the micro-array chip can utilize cDNA, amplified production
Expression degree and control group be compared confirmations, and be able to confirm that to less than 2.5 microns fine dusts specificity mistake tables
Up to or low expression gene distribution such that it is able to usefully as being confirmed whether exposed to less than 2.5 microns fine dusts, 2.5
The monitoring of the following fine dust of micron, the harm sex determination of less than 2.5 microns fine dusts and find out because less than 2.5 microns thin
The purposes of the toxic effect mechanism that micro- dust causes.
Below according to embodiment and experimental example, the present invention is described in detail.
But, following embodiments and experimental example are served only for illustrating the present invention, and non-invention is by following embodiments
And experimental example is limited.
<Embodiment 1>Less than 2.5 microns fine dust trappings and extraction
<1-1>Selected trapping place
In order to excavate to the gene expression exposed to PM2.5 whether confirmation biomarker, positioned at Shouer City Chengbei District
The Korean Institute of Science and Technology gymnasium roof in lower valley hole be chosen to be test portion trapping place, trapped 2.5 microns (μm) with
Lower fine dust, the lower valley hole abuts with the inner ring road near the valley station of Chengbei District, and the road in November, 2011 usually puts down
Equal more than 70,000, December usually it is average more than 6.9 ten thousand process, possess the suitable volume of traffic.In order to implement to check, it is necessary to substantial amounts of
Less than 2.5 microns fine dusts, thus low volume air sampler (Low Volume Air Sampler) start 24 hours when
Less than the 2.5 microns amounts of fine dust being obtained in that only several milligrams of (㎎), needed for not being suitable for being checked in experiment tube
The trapping of less than 2.5 microns fine dusts, so the high flow capacity air sampler that can largely trap for one day of selection has carried out reality
Test.For less than the 2.5 microns special Inlet of fine dust (Tisch instrument companies), aerodynamic size
(Aerodynamic Size) is more than less than 2.5 microns fine dust persons, it is impossible to defeat the inertia of its weight, collides oil film
(oil) stop, being carried out using polytetrafluorethylecoatings coatings glass fiber filter paper by less than 2.5 microns fine dusts of the process
Trapping.
<1-2>Trapping filter paper
Polytetrafluorethylecoatings coatings glass fiber filter paper, the polytetrafluoroethylene (PTFE) are employed during using high flow capacity air sampler
Coating glass fiber filter paper is this have the advantage that in filtration step, without 8 × 10 inches of compositions of (inch) size of extraction
Possibility, as Extraction solvent, make use of purified water (distilled water, 18.2M Ω) and dichloromethane
(dichloromethane).In order that because of error realization minimum caused by moisture, passing through in drier (decicator)
48 hours, after the moisture of filter paper is removed completely, utilization can be measured to the ultraprecise weighing device of 0.1mg (plum Teller-Tuo Li
Many companies), weigh the weight of filter paper.The medial surface of filter paper it is intersecting fold after, be put into darkroom and obturation effect
Zippered bag (Zipper bag), until before using test portion, taken care of at -80 DEG C, when trapping place is moved to, using ice
Case (Ice box) is moved.For less than 2.5 microns fine dust trappings, appropriate flow speed (1.13m is kept3/min±
10%).
<1-3>During trapping
Less than 2.5 microns fine dusts were trapped after 5 months using high flow capacity air sampler.2011 11
Month 1 day start to trap for the first time, last 50th trapping of the end of day of March 28 in 2012.When rainy, atmospheric concentration is being represented
Filter paper value in, the μ g/m of value 20 on the basis of measured value3Following value is excluded, and is grouped by each 10 filter paper, and 5 are constituted altogether
Group.
<1-4>Fine dust traps test portion abstraction process
For less than the 2.5 microns constituent analyses of fine dust of trapping, a filter paper is divided into 1/2 and is used, as mutual
The two kinds of Extraction solvents for differing, each filter paper make use of the super clean for water-soluble (water soluble) constituents extraction
Change water (distilled water) and the dichloromethane for Organic (organic soluble) constituents extraction.As shown in figure 1, according to carrying
Operation path is taken, less than 2.5 microns fine dusts water solubilitys and 2.5 Organic extract solutions (Fig. 1) have been made respectively.
<Embodiment 2>Selecting for PM2.5 exposure models and receiving for mouse lung tissue test portion
<2-1>PM2.5 exposure models
It is suspended solid in granular air as the PM2.5 of environmentally hazardous substance.Particulate material have enter lung
Tendency that is interior and inducing typicalness particle induction inflammation (particle induced inflammation).As for sensitive
Ground is measured the animal model of this reaction and has largely been reported using the research of BALB/C, due to the original in BALB/C characteristics
Cause, the feature with easy infection chronic pneumonia, it is contemplated that, relatively docile, breast carcinogenesis ratio very high to the sensitiveness of radioactive ray
The low characteristic of rate, finally have selected the suitable mouse model.
Specifically, as shown in table 1, described<Embodiment 1>The PM2.5 of middle trapping, is divided into two different kinds of feature
The water solubility (Water soluble) of class and Organic (ganic soluble) group, are divided into exposure group and non-exposed group,
And exposed to mouse model, in addition, make exposure group exposed to low concentration PM2.5 (low capacity) and high concentration PM2.5 (high power capacity),
It is carried out (table 1) including including non-exposed group, being divided into 6 groups.
【Table 1】
PM2.5 exposure concentrations and mouse experiment group are constituted
| Experimental group | Exposed amount (absolute magnitude) | Number of individuals (BALB/C) |
| Control group | 0 | 24 |
| Low capacity | 20 | 24 |
| High power capacity | 164 | 24 |
<2-2>The selected and exposure of the PM2.5 exposure concentrations in mouse model
In order to select the PM2.5 exposure concentrations in mouse model, the research method and knot of the document of report before with reference to
Really (Hans Hedrich.The laboratory mouse, 2004), it is contemplated that occur in the fatal rate of mouse and actual environment
The distributed density of PM2.5 etc., select control group (0g), low capacity (low dose) (20g), high power capacity (high dose)
After three kinds of concentration of (164g), exposure experiment (table 1) has been carried out.
In process for exposing, for PM2.5 test portions, although being actually suitably executed suction experiment, caused when actual
During suction, it is impossible to by required concentration, in the same manner exposed to multiple individual mouse, thus two groups of PM2.5 manufactures of trapping
Into the test portion of liquid condition, (tracheal instillation) mode is directly administered by tracheal strips, with following calculating sides
Method, BALB/C mice model is exposed to by group.
PM2.5 mouse dosage computational methods
When 20 μ g are administered
The μ g/m3 of daily atmospheric environment benchmark 50 of PM2.5
When exposing 1 time-of-week by 25 μ g~50 μ g
Therefore, when PM2.5 is exposed to mouse by 20 μ g, it isWhen with the air PM2.5
Environmental criteria is applied to the exposed amount during mankindWhen comparing, it is judged as applicable exposed amount.
Carry out the low concentration (low dose) expose the reasons why because, although there is not cytotoxicity, histopathology
Effect, but do not know whether the change of gene level occurs.In contrast, in the case of 164g, it is expected to cell occur
Toxicity, the effect of histopathology, in addition, being expected to the change of gene level also occur, thus are set to high concentration exposed amount.
<2-3>Lung tissue test portion
In BALB/C mice model, for the purpose that high-quality total serum IgE is preferentially extracted from target organ, thus maximum limit
Ensure that the lung tissue of non-faulted condition degree.After sampling, using can take care of under -20 DEG C and shading status 6 months, -
Full guard solution (the Allprotect of the fast and smart company (Qiagen) of 1 year can be taken care of under 80 DEG C and shading status
Solution), before the use, taken care of at -80 DEG C.
<Embodiment 3>Microarray Experiments
<3-1>The separation of target RNA and fluorescent material are marked
RNA is extracted from PM2.5 exposures group and non-exposed group blood test portion, is to utilize(Life Technologies, Inc., it is beautiful
State), extract skill and technique using the RNA of manufacturer and extracted, the mini reagents of RNeasy of smart company (Qiagen) using fast
Box (RNeasy Mini kit) (Cat NO.74106) is purified.Genomic DNA uses nothing during RNA is purified
DNase suits (RNase-free DNase set) (Qiagen, USA) of RNase is removed.Each total serum IgE test portion it is dense
Spend by the use of NanoDrop ND 1000 spectrophotometer (the spectrophotometer) (NanoDrop as spectrophotometer
Technology company, the U.S.) measured, the state of the matter of RNA utilizes the biological analyser (Agilent of Agilent 2100
2100Bioanalyzer) measured.
<3-2>The manufacture of the cDNA of mark
For oligosaccharides microarray analysis, using in the embodiment<2-1>Middle less than the 2.5 microns fine dust water received
The total serum IgE of dissolubility extract solution, Organic extract solution treatment group, has manufactured cDNA.The above-mentioned μ g of total serum IgE 30 and oligosaccharides for receiving
(dT) primerMixing, after 10 minutes are reacted at 65 DEG C, is immediately placed in annealing in ice
(annealing).In order to the reverse transcription (Reverse Transcript) of the RNA of the annealing reacts, shown in following [table 2]
Mix reagent.Now, two kinds of test portions mix and purified using Microcon YM-30 pillars (Millipore, the U.S.).
【Table 2】
<3-3>Hybridization reaction
Hybridization and washing process are performed by the reminding method of (strain) Biogen.
Specifically, it is as shown in table 3 below, prepare transcription premix buffer solution (Transcription Master Mix) and put
Enter, after being sufficiently mixed, 2 hours are reacted at 40 DEG C, then carry out the purification of the cRNA to labeled (Labeling)
(purification) after process, the cRNA 600ng of purified process are reacted 30 minutes at 60 DEG C, performs fracture
(Fragmentation) process.2 × GEx Hybridization Buffer HI- are put into the cRNA for preparing as described above
After RPM and mixing, chip (chip) is put in, (hybridization) 17 hours at 65 DEG C, are hybridized in an oven.
After 17 hours, (carried out in GE Wash Buffer 11 minute, carried out in GE Wash Buffer2 by washing process
1 minute) after, the chip has been dried 3 minutes with 800rpm centrifugations.
【Table 3】
<3-4>Obtain fluoroscopic image
Hybridization image Agilent C-scan instrument (Agilent C scanner) (Agilent Technologies, U.S. on slide glass
State) scanned, data separate Agilent GeneSpring GX12.6.1 (the Agilent GeneSpring GX of extraction
12.6.1) (Agilent Technologies), by normalization (normalization), analyze the expression pattern of each gene.
Its result is as shown in Figure 2, it is thus identified that present on oligosaccharides chip about 55, in more than 681 gene, exposure group with
There is different (Fig. 2), the result checked and approved this, as shown in table 4 and Fig. 3 in the gene expression pattern of non-exposed group, it is thus identified that
Expression increases by more than 1.5 times of 25 kinds of genes (table 4 and Fig. 3).
【Table 4】
Because expressing the gene for changing exposed to PM2.5
Claims (9)
1. 2.5 microns (μm) of a kind of gene being integrated with more than a certain kind selected from following groups or its complementary strand molecule is thin below
Whether micro- dust (particulate matter 2.5, PM2.5) exposes and confirms with micro-array chip (microarray
chip)：
Gene accession number (Genebank) NM_001163145 (1810041L15Rik, RIKEN cDNA
1810041L15gene), gene accession number (Genebank) NM_001081381 (2610002I17Rik, RIKEN cDNA
2610002I17), gene accession number (Genebank) NM_176952 (6430573F11Rik, RIKEN cDNA 6430573F11
Gene), gene accession number (Genebank) AK141429 (BC023202, cDNA sequence BC023202), gene registration
Number (Genebank) NM_144890 (Bpifb5, BPI fold containing family B, member 5), gene registration
Number (Genebank) NM_178626 (Cdc42se2, CDC42 small effector 2), gene accession number (Genebank)
NM_199032 (Cep135, centrosomal protein 135), gene accession number (Genebank) NM_146023
(Evi2b, ecotropic viral integration site 2b), gene accession number (Genebank) AK146791
(Ewsr1, Ewing sarcoma breakpoint region 1), gene accession number (Genebank) NM_197989
(Fam58b, family with sequence similarity 58, member B), gene accession number (Genebank) NM_
010200 (Fgf13, fibroblast growth factor 13), gene accession number (Genebank) NR_003248
(Foxl2os, forkhead box L2 opposite strand transcript), gene accession number (Genebank) NR_
037689 (Gm1968, predicted gene 1968), gene accession number (Genebank) XM_001472451 (Gm2102,
Predicted gene 2102), gene accession number (Genebank) AK078977 (Mapkapk3, mitogen-activated
Protein kinase-activated protein kinase 3), gene accession number (Genebank) NM_001037906
(Nell1, NEL-like 1 (chicken)), gene accession number (Genebank) NM_001134480 (Plcxd2,
Phosphatidylinositol-specific phospholipase C, X domain containing 2), gene registration
Number (Genebank) NM_008895 (Pomc, pro-opiomelanocortin-alpha), gene accession number (Genebank)
NM_011198 (Ptgs2, prostaglandin-endoperoxide synthase 2), gene accession number (Genebank)
NM_028762 (Rbm19, RNA binding motif protein 19), gene accession number (Genebank) NM_033354
(Sec16b, SEC16 homolog B (S.cerevisiae)), gene accession number (Genebank) NM_001254731
(Sgsm1, small G protein signaling modulator 1), gene accession number (Genebank) NM_199199
(Tmem199, transmembrane protein 199), gene accession number (Genebank) NM_026454 (Ube2f,
Ubiquitin-conjugating enzyme E2F (putative)) and gene accession number (Genebank) NM_026615
(Urm1,ubiquitin related modifier 1homolog(S.cerevisiae))。
2. whether less than 2.5 microns fine dusts according to claim 1 expose and confirm to use micro-array chip, and its feature exists
In described 2.5 microns of (μm) following fine dusts are less than the 2.5 microns water-soluble or Organic extract solutions of fine dust.
3. whether a kind of 2.5 microns (μm) following fine dust of micro-array chip comprising described in claim 1 exposes and confirms
Use kit.
4. whether less than 2.5 microns fine dusts according to claim 3 expose and confirm to use kit, it is characterised in that
It is additional to include mouse pneumonocyte or lung tissue.
5. it is a kind of comprising can with following gene complementations, it is sudden and violent to less than 2.5 microns fine dusts of the primer of following gene magnifications
Whether reveal and confirm to use kit：
Gene accession number (Genebank) NM_001163145 (1810041L15Rik, RIKEN cDNA 1810041L15
Gene), gene accession number (Genebank) NM_001081381 (2610002I17Rik, RIKEN cDNA 2610002I17),
Gene accession number (Genebank) NM_176952 (6430573F11Rik, RIKEN cDNA 6430573F11 gene), gene
Registration number (Genebank) AK141429 (BC023202, cDNA sequence BC023202), gene accession number
(Genebank) NM_144890 (Bpifb5, BPI fold containing family B, member 5), gene accession number
(Genebank) NM_178626 (Cdc42se2, CDC42 small effector 2), gene accession number (Genebank) NM_
199032 (Cep135, centrosomal protein 135), gene accession number (Genebank) NM_146023 (Evi2b,
Ecotropic viral integration site 2b), gene accession number (Genebank) AK146791 (Ewsr1, Ewing
Sarcoma breakpoint region 1), gene accession number (Genebank) NM_197989 (Fam58b, family with
Sequence similarity 58, member B), gene accession number (Genebank) NM_010200 (Fgf13,
Fibroblast growth factor 13), gene accession number (Genebank) NR_003248 (Foxl2os, forkhead
Box L2opposite strand transcript), gene accession number (Genebank) NR_037689 (Gm1968,
Predicted gene 1968), gene accession number (Genebank) XM_001472451 (Gm2102, predicted gene
2102), gene accession number (Genebank) AK078977 (Mapkapk3, mitogen-activated protein kinase-
Activated protein kinase 3), gene accession number (Genebank) NM_001037906 (Nell1, NEL-like 1
(chicken)), gene accession number (Genebank) NM_001134480 (Plcxd2, phosphatidylinositol-
Specific phospholipase C, X domain containing 2), gene accession number (Genebank) NM_008895
(Pomc, pro-opiomelanocortin-alpha), gene accession number (Genebank) NM_011198 (Ptgs2,
Prostaglandin-endoperoxide synthase 2), gene accession number (Genebank) NM_028762 (Rbm19,
RNA binding motif protein 19), gene accession number (Genebank) NM_033354 (Sec16b, SEC16
Homolog B (S.cerevisiae)), gene accession number (Genebank) NM_001254731 (Sgsm1, small G
Protein signaling modulator 1), gene accession number (Genebank) NM_199199 (Tmem199,
Transmembrane protein 199), gene accession number (Genebank) NM_026454 (Ube2f, ubiquitin-
Conjugating enzyme E2F (putative)) and gene accession number (Genebank) NM_026615 (Urm1,
ubiquitin related modifier 1homolog(S.cerevisiae))。
6. whether a kind of less than 2.5 microns fine dusts expose confirmation method, it is characterised in that including：
1) in the body cell for from the body cell for as a corpse or other object for laboratory examination and chemical testing for experimental group separate and from normal individual as a control group separate
The step of separating each RNA；
2) step 1) experimental group and the RNA of control group while synthesize cDNA, used each different fluorescence
The step of matter is marked；
3) make step 2) with fluorescent material mark cDNA with DNA microarray chip hybridization of the invention the step of；
4) analytical procedure 3) reaction micro-array chip the step of；And
5) in step 4) analysis data in, the table of the gene of the micro-array chip for being integrated in claim 1 of experimental group
The step of confirmation being compared up to degree and control group.
7. whether less than 2.5 microns fine dusts according to claim 6 expose confirmation method, it is characterised in that
Step 1) body cell for mouse pneumonocyte or lung tissue.
8. whether less than 2.5 microns fine dusts according to claim 7 expose confirmation method, it is characterised in that
The lung tissue is BALB/C mice lung tissue.
9. whether less than 2.5 microns fine dusts according to claim 6 expose confirmation method, it is characterised in that
Step 2) fluorescent material by Cy3 (cyanine 3), Cy5, poly-l-lysine fluorescein isothiocynate (poly L-
Lysine-fluorescein isothiocyanate, FITC), rhodamine B-isothiocyanate (rhodamine-B-
Isothiocyanate, RITC) and rhodamine (rhodamine) constitute group in select to use.
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| KR102255624B1 (en) | 2020-09-03 | 2021-05-25 | 한국화학연구원 | Method for analyzing content ratio of pm2.5 and pm10 of fine particle |
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