CN104878088A - Indium-exposed molecular marker - Google Patents
Indium-exposed molecular marker Download PDFInfo
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- CN104878088A CN104878088A CN201510204378.8A CN201510204378A CN104878088A CN 104878088 A CN104878088 A CN 104878088A CN 201510204378 A CN201510204378 A CN 201510204378A CN 104878088 A CN104878088 A CN 104878088A
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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
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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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
- C12Q2600/142—Toxicological screening, e.g. expression profiles which identify toxicity
Abstract
The invention discloses an indium-exposed molecular marker. The invention provides application of indium or compounds indium in promoting oxidative damage of mitochondrions, and application of the indium or compounds thereof in preparing products for promoting oxidative damage of mitochondrions. The experiment proves that after indium chloride is added into in-vitro blood and cultured, the changes of the blood cell mitochondrions are different from those of normal cells, which indicates that the indium chloride can enhance the relative content of the mitochondrion ND1 gene in cells. Thus, the mitochondrions are subjected to oxidative damage in the indium exposure process, and the mitochondrion quantitative detection can be used as a potential biomarker for hazards of exposure of indium and compounds thereof.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of indium and expose molecular marker.
Background technology
Indium (indium, In) is one of necessary raw material of liquid crystal display, LED and semiconductor manufacturing, along with global electronics industry to develop its demand rapidly increasing.Since Japanese scholars report indium in 2003 exposes poisoner, cause the great attention of each production power, China and the U.S. also report occupational indium and expose poisoner.On December 23rd, 2013, national health State Family Planning Commission, Administration of Security Supervision, manpower Department of Social Security and National Federation of Trade Unions's combination weave have printed and distributed China's " occupational illness classification and catalogue ", and wherein, to be increased to occupational chemical poisoning by poisoning for Indium and compounds.Kazuyuki Omae etc. has carried out epidemiological investigation to this new occupational indium tuberculosis.The case report of the pulmonary disorder using computer search to be correlated with about indium and epidemiological study.Result is by March, 2010, and 7 routine interstitial pneumonias occur Japanese indium operating worker, and 2 routine pulmonary alveolar proteinosises (PAP) occur U.S.'s indium Exposure, and it is poisoning that 1 routine indium occurs Chinese indium Exposure, and the cross-section survey of 4 routine patients to Japan.Show in Japan is to all cases and epidemiological study, be exposed to insoluble indium compound and can cause interstitial pneumonia and pulmonary emphysema, definition be " indium lung ".Also combine restriction KL-6 increasing degree based on epidemiological study result, Japanese Occupational health association advises that 3 micrograms per litre serum indiums are as professional exposure limit.Research is thought current to long term follow-up and was once engaged in indium operating worker and is very important, and not only will understand the natural history of indium lung, and will follow the tracks of the incidence of lung cancer.Irreversible after the morbidity of above-mentioned place case, had 3 examples dead, therefore avoided occupational hazards, it is very necessary to probe into its pathogenic factor.Indium principal causative mechanism is completely not clear and definite, and large quantifier elimination finds what the poisoning mainly oxidative damage of Indium and compounds caused before.Indium and compounds can produce toxicity and bring out alveolar cell and phagocytic cell paraplasm, and mechanism is mainly passed through modes such as mitochondrial oxidative stress, DNA damage, acceleration apoptosis.Chinese scholars, to plastosome and poisoning being studied of Indium and compounds, is mainly gathered in the research of serum protein markers, but there are no for the poisoning important organelle-mitochondrial research causing oxidative damage of Indium and compounds.
ND1 gene is the most conservative in chondriogen, and its expression product is nadh dehydrogenase (complex body I) subunit 1.The change of chondriogen content may react exogenous material have activated cellular oxidation stress process.The increase of chondriogen content can produce the impact of two aspects.On the one hand, it stimulates mitochondrial hyperplasia to provide energy to meet the necessity of cells survival, comprises and repairs damage and synthesize new protein.On the other hand, the mitochondrial function imbalance become increasingly abundant causes active oxygen surplus to be produced and further oxidative damage, and possible active cell is old and feeble or dead, and then causes more serious pathological consequences.
Summary of the invention
An object of the present invention is to provide the application of indium or its compound.
The invention provides indium or the application of its compound in promotion Mitochondrial oxidative damage.
Or,
The invention provides indium or the application of its compound in preparation promotion Mitochondrial oxidative damage product.
In above-mentioned application, described promotion Mitochondrial oxidative damage is embodied in and improves mitochondrial ND1 gene gene relative content.
In above-mentioned application, described mitochondrial ND1 gene gene relative content is the value that mitochondrial ND1 gene CT value-HGB1 gene C T value obtains.
In above-mentioned application, described mitochondrial ND1 gene CT value is obtained by the RT-PCR amplification of primer pair 1, and described primer pair 1 is made up of the single strand dna shown in sequence 2 in the single strand dna shown in sequence in sequence table 1 and sequence table;
Described Mitochondrial H GB1 gene C T value is obtained by the RT-PCR amplification of primer pair 2, and described primer pair 2 is made up of the single strand dna shown in sequence 4 in the single strand dna shown in sequence in sequence table 3 and sequence table.
In above-mentioned application, described plastosome derives from isolated cells, is specially isolated blood cell.
Another object of the present invention is to provide another purposes of indium or its compound.
The invention provides indium or the application of its compound in raising mitochondrial ND1 gene gene relative content;
Or,
Indium or its compound improve the application in mitochondrial ND1 gene gene relative content product in preparation.
In above-mentioned application, described mitochondrial ND1 gene gene relative content is the value that mitochondrial ND1 gene CT value-HGB1 gene C T value obtains.
In above-mentioned application, described mitochondrial ND1 gene CT value is obtained by the RT-PCR amplification of primer pair 1, and described primer pair 1 is made up of the single strand dna shown in sequence 2 in the single strand dna shown in sequence in sequence table 1 and sequence table;
Described Mitochondrial H GB1 gene C T value is obtained by the RT-PCR amplification of primer pair 2, and described primer pair 2 is made up of the single strand dna shown in sequence 4 in the single strand dna shown in sequence in sequence table 3 and sequence table.
In above-mentioned application, described plastosome derives from isolated cells, is specially isolated blood cell.
In above-mentioned application, described indium compound is indium chloride.
The application of material in preparation diagnosis or the auxiliary diagnosis body product whether indium exposes or indium is poisoning of detection line plastochondria whether oxidative damage is also the scope of protection of the invention;
Or,
The application of the material that in detection line plastochondria, whether ND1 gene content improves in preparation diagnosis or the auxiliary diagnosis body product whether indium exposes or indium is poisoning is also the scope of protection of the invention.
In above-mentioned application, above-mentioned substance comprises indium or its compound, and above-mentioned substance also comprises primer pair 1 and primer pair 2.
Experiment of the present invention proves, the present invention cultivates by adding indium chloride in extracorporeal blood, and the mitochondrial change of its blood cell and normal cell there are differences, and finds that indium chloride can improve cell Mitochondria ND1 gene relative content.Illustrate that exposing process Mitochondria at indium there occurs oxidative damage, plastosome detection by quantitative can be used as the potential source biomolecule mark that Indium and compounds exposes harm.
Accompanying drawing explanation
Fig. 1 is fluorescent PCR amplification curve diagram.
Fig. 2 is solubility curve figure.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain (yes) from commercial channels.
In following embodiment, instrument equipment is as follows: American AB I7300 fluorescent PCR amplification instrument, adjustable quantitative liquid feeder, spirit lamp, autoclave sterilization apparatus, Bechtop, analytical balance and common drug balance, culturing bottle, graduated centrifuge tube (5ml, 10ml), heparin sodium anticoagulant tube, disposable plastic tube, water isolation type constant incubator (outfit temperature indicating controller (TIC)), Constant Temp. Oven, electric centrifuge (horizontal)
In following embodiment, main matched reagent used is as follows: DNA extraction reagent extracts reagent purchased from the clean husky serial pillar blood DNA in sky, Beijing bounties genome company sky, Fluorescence PCR reagent is purchased from Dalian precious biotech firm RR820A Fluorescence PCR reagent, and other biochemical reagents and primed probe synthetics are purchased from Shanghai Sheng Gong bio-engineering corporation.Modified form RPMI-1640 substratum, foetal calf serum, PHA (phytoh(a)emagglutinin), distilled water, injection heparin sodium.
The cleaning of Glass tubing ware and sterilization in following embodiment: first with washing powder, vessel are washed, tap water dries, then put in washings and soak 1-2 days, vessel through soaking rush 15-20 minute with tap water after taking out, then shake with waterside trimming from the beginning and wash 5 times, 3 times are rushed again, drying 2 hours at 100-120 DEG C with distilled water.
The autoclave sterilization of vessel in following embodiment: preparation substratum vessel used, after cleaning, drying, are wrapped with infantees and made autoclaving sterilization (15 pounds 20 minutes), take out dry for standby.
Embodiment 1, indium are improving the application in chondriogen ND1 gene relative content
1, cell culture medium preparation
Basic medium configures in accordance with the following steps: PHA (phytohaemagglutinin), heparin sodium, gentamicin sulphate, glutamine and RPMI-1640 nutrient solution are mixed, obtain basic medium, wherein, the final concentration of PHA is 160 μ g/mL, the final concentration of heparin sodium is 37.5IU/ml, the final concentration of gentamicin sulphate is 112IU/ml, the final concentration of glutamine is 0.03%.
Growth medium configures in accordance with the following steps: newborn calf serum, foetal calf serum and basic medium are mixed, obtain growth medium, wherein, the final concentration of calf serum is 10% (volumn concentration), the final concentration of foetal calf serum is 10% (volumn concentration).
2, blood sampling is cultivated
Extract 15 normal, disease-free crowds (patient knows the inside story) venous blood 5ml, in heparin sodium anticoagulant tube, fully to mix, immediately censorship, obtain 15 anticoagulation samples.
3, cell cultures
Draw 1500ul at each sample of clean bench continuous and quantitative pipettor, each sample is divided into three groups:
0mmol/L indium chloride group (Normal group): 500ul is placed in culturing bottle, adds 3.0ml growth medium;
0.2mmol/L indium chloride group (0.2mmol/L indium chloride exposed cell culture group): 500ul is placed in culturing bottle, adds 3.0ml growth medium, and to add indium chloride to final concentration be 0.2mmol/L;
0.8mmol/L indium chloride group (0.8mmol/L indium chloride exposed cell culture group): 500ul is placed in culturing bottle, adds 3.0ml growth medium, and to add indium chloride to final concentration be 0.8mmol/L;
Above-mentioned three groups of culturing bottles are put the CO of 37.5 ± 0.5 DEG C
2cultivate 3 days in incubator.
4, detection line mitochondrial genes relative content
1) extraction of cell DNA
In three groups of culturing bottles, 100ul cell culture fluid within 3rd day, is drawn for extracting cell DNA respectively what cultivate.
DNA extraction reagent extracts reagent purchased from the clean husky serial pillar blood DNA in sky, Beijing bounties genome company sky, and concrete extracting method is as follows:
The cell culture fluid of 100uL mixing is added in 1.5ml centrifuge tube, in the centrifuge tube of 100uL cell culture fluid, adding 0.5mL solution A, (solution A has a small amount of crystal settling, rock evenly with front needs), with liquid-transfering gun piping and druming, precipitation is fully suspended, this step object is lysing cell, released dna, so piping and druming is more fully better.Liquid rotating moves on in centrifugal adsorbing column and also leaves standstill 2-5 minute, be fully combined to make DNA with film by concussion mixing for 30 minutes after solution change is limpid.12,000rpm centrifugal half a minute, DNA will be adsorbed onto on film, abandon the waste liquid in collection tube.Add the general of 0.5mL and wash post liquid, 12,000rpm centrifugal half a minute, abandon the waste liquid in collection tube.Add the general of 0.5mL and wash post liquid, 12,000rpm centrifugal half a minute, abandon the waste liquid in collection tube.12,000rpm centrifugal half a minute, dry residual liquid.This step is very important, residual generally washes post liquid, otherwise can affect subsequent reactions to remove on film.Centrifugal adsorbing column is placed in a new 1.5mL plastic centrifuge tube (providing for oneself), add the general elutriant of appropriate 150uL, room temperature places 2 minutes.Re-used after 65 DEG C of preheatings by general elutriant, the effect of eluted dna can be better.12,000rpm centrifugal half a minute, solution at the bottom of centrifuge tube and DNA.Can use immediately or put refrigerator and preserve for a long time.
2) RT-PCR amplification
With the HGB1 gene primer shown in table 1 to ND1 gene primer to carrying out RT-PCR amplification to the genomic dna of said extracted respectively.
Table 1 detects primer sequence
RT-PCR reaction system adopts Dalian precious biotech firm RR820A fluorescence RT PCR reaction reagent, and cumulative volume 20 μ l, is specially:
premix Ex TaqII (Tli RNaseH Plus) (2 ×) 10.0 μ l, upstream primer (10 μMs) 0.8 μ l, downstream primer (10 μMs) 0.8 μ l, ROX Reference Dye (50 ×) 0.4 μ l, DNA profiling 2.0 μ, dH
2o (sterile purified water) 6.0 μ l.
Annealing temperature presses 1 DEG C, 55-60 DEG C of interval assay optimization respectively, when annealing temperature is 56 degree, response curve is typical S type, and fluorescent signal is the strongest, this experimental selection 56 degree carries out all Fluorescence PCRs below as annealing temperature, concrete RT-PCR reaction conditions: 95 DEG C of 10s; 95 DEG C of 5s, 56 DEG C of 34s, 40Cycles.
3) detect
The program making amplification curve diagram utilizing PCR instrument to carry, after reaction terminates, draws solubility curve immediately.
Many groups mean compares employing variance analysis test, and P<0.05 has significant difference.All data statistic analysis all use SPSS13.0 software analysis.
Quantitative PCR reacts, the program making amplification curve diagram (Fig. 1) utilizing PCR instrument to carry, and after reaction terminates, draws solubility curve (Fig. 2) immediately.
Along with PCR system measures absorbance after each loop ends, the coordinate curve that just to obtain with cycle number be X-coordinate, absorbance is ordinate zou, detecting cycle index flex point corresponding to of sample from baseline to exponential growth is Ct value, and chondriogen relative content is ND1 gene C T value-HGB1 gene C T value.
Result is as shown in table 2.
Table 2 is fluorescent quantitative PCR CT value
AOVA variance analysis, statistical study three is group chondriogen gene relative content mean total difference respectively.
0.8umol/L indium chloride exposed cell culture group, 0.2umol/L indium chloride exposed cell culture group and be normally quantitatively respectively-4.49 ± 0.39 ,-5.02 ± 0.51 and-4.99 ± 0.43 without exposed cell culture group chondriogen gene relative content.Wherein after the inoculation of 0.8mmol/L indium chloride exposed cell culture group, the mean of the 3rd day CT value difference value is the highest, higher than Normal group and 0.2mmol/L indium chloride exposed cell culture group.
TTEST statistics display 0.8mmol/ indium chloride exposed cell culture group compares, specifically in table 3 with the mean of 0mmol/L indium chloride Normal group CT value difference value.
Table 3 is comparing of increasing with infection time Peripheral Blood cell mitochondrial of the indium chloride of different concns
Can find out, 0.8mmol/L indium chloride exposed cell culture group compares with control group, P < 0.005.0.8mmol/L indium chloride exposed cell culture group compares 2P < 0.005 with 0.2mmol/L indium chloride exposed cell culture group, can find out, 0.8mmol/L group is compared with other groups, chondriogen ND1 gene relative content difference has statistical significance (P<0.005), show, plastosome sustains damage.
Claims (10)
1. indium or its compound are promoting the application in Mitochondrial oxidative damage;
Or,
Indium or its compound promote the application in Mitochondrial oxidative damage product in preparation.
2. application according to claim 1, is characterized in that: described promotion Mitochondrial oxidative damage is embodied in and improves mitochondrial ND1 gene gene relative content.
3. application according to claim 1 and 2, is characterized in that: described mitochondrial ND1 gene gene relative content is the value that mitochondrial ND1 gene CT value-HGB1 gene C T value obtains.
4. according to described application arbitrary in claim 1-3, it is characterized in that: described mitochondrial ND1 gene CT value is obtained by the RT-PCR amplification of primer pair 1, and described primer pair 1 is made up of the single strand dna shown in sequence 2 in the single strand dna shown in sequence in sequence table 1 and sequence table;
Described Mitochondrial H GB1 gene C T value is obtained by the RT-PCR amplification of primer pair 2, and described primer pair 2 is made up of the single strand dna shown in sequence 4 in the single strand dna shown in sequence in sequence table 3 and sequence table.
5., according to described application arbitrary in claim 1-4, it is characterized in that:
Described plastosome derives from isolated cells, is specially isolated blood cell.
6. indium or its compound are improving the application in mitochondrial ND1 gene gene relative content;
Or,
Indium or its compound improve the application in mitochondrial ND1 gene gene relative content product in preparation.
7. application according to claim 6, is characterized in that: described mitochondrial ND1 gene gene relative content is the value that mitochondrial ND1 gene CT value-HGB1 gene C T value obtains.
8. the application according to claim 6 or 7, it is characterized in that: described mitochondrial ND1 gene CT value is obtained by the RT-PCR amplification of primer pair 1, and described primer pair 1 is made up of the single strand dna shown in sequence 2 in the single strand dna shown in sequence in sequence table 1 and sequence table;
Described Mitochondrial H GB1 gene C T value is obtained by the RT-PCR amplification of primer pair 2, and described primer pair 2 is made up of the single strand dna shown in sequence 4 in the single strand dna shown in sequence in sequence table 3 and sequence table.
9., according to described application arbitrary in claim 6-8, it is characterized in that: described plastosome derives from isolated cells, be specially isolated blood cell;
Described indium compound is indium chloride.
10. the application of material in preparation diagnosis or the auxiliary diagnosis body product whether indium exposes or indium is poisoning of detection line plastochondria whether oxidative damage;
Or,
The application of the material that in detection line plastochondria, whether ND1 gene content improves in preparation diagnosis or the auxiliary diagnosis body product whether indium exposes or indium is poisoning.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006138145A1 (en) * | 2005-06-14 | 2006-12-28 | Northwestern University | Nucleic acid functionalized nanoparticles for therapeutic applications |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006138145A1 (en) * | 2005-06-14 | 2006-12-28 | Northwestern University | Nucleic acid functionalized nanoparticles for therapeutic applications |
Non-Patent Citations (3)
| Title |
|---|
| LIN RH: "Indium Chloride-Induced Micronuclei via Reactive Oxygen Species in Chinese Hamster Lung Fibroblast V79 Cells", 《ENVIRONMENTAL TOXICOLOGY》 * |
| 彭琼玲: "还原型烟酰胺腺嘌呤二核苷酸脱氢酶在早产大鼠高氧肺损伤后肺组织中的动态表达及其意义", 《中华医学会第十四次全国儿科学术会议论文汇编》 * |
| 赵静珺: "硫酸铟对 V79 细胞内活性氧和线粒体膜电位的影响", 《环境与职业医学》 * |
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