A kind of molecular marker of indium exposureTechnical field
The present invention relates to biological technical field, more particularly to a kind of indium exposure molecular marker.
Background technology
Indium (indium, In) is one of necessary raw material of liquid crystal display, LED light and semiconductor manufacturing, with global electronic workIndustry to develop its demand rapidly increasing.Since Japanese scholars report indium exposure poisoner in 2003, each system has been causedThe great attention of industry power is made, China and the U.S. also report occupational indium exposure poisoner.On December 23rd, 2013, country defendsRaw State Family Planning Commission, Administration of Security Supervision, human resources Department of Social Security and National Federation of Trade Unions's united organization have printed and distributed China《Occupational diseaseClassification and catalogue》, wherein, Indium and compounds poisoning is increased into occupational chemical poisoning.Kazuyuki Omae etc. are to thisNew occupational indium tuberculosis has carried out epidemiological investigation.With disease of the computer search on the relevant pulmonary disease of indiumExample report and epidemiological study.As a result 7 interstitial pneumonias, U.S.'s indium occurs by March, 2010, Japanese indium operating worker2 pulmonary alveolar proteinosises (PAP) occur for Exposure, and 1 indium poisoning occurs for Chinese indium Exposure, and to 4 of JapanThe cross-section survey of patient.Show in Japan is to all cases and epidemiological study, can exposed to slightly solubility indium compoundCause interstitial pneumonia and pulmonary emphysema, definition is " indium lung ".Based on epidemiological study result and combine limitation KL-6 increasingsAdd amplitude, Japanese occupational health association suggests 3 micrograms per litre serum indiums as professional exposure limit.Research is thought to long term follow-upIt is current and be once engaged in indium operating worker and be very important, not only it is to be understood that the natural history of indium lung, but also to track lungThe incidence of cancer.It is irreversible after above-mentioned place case morbidity, there are 3 death, therefore occupational hazards is avoided, probe into its hairCause of disease is because very necessary.Not completely clearly, substantial amounts of research before finds Indium and compounds poisoning master to indium principal causative mechanismIf caused by oxidative damage.Indium and compounds can produce toxicity and induce alveolar cell and phagocyte paraplasm, machineReason is mainly by modes such as the oxidative stress of mitochondria, DNA damage, acceleration Apoptosis.Domestic and foreign scholars are to mitochondriaWith Indium and compounds poisoning study, be mainly gathered in the research of serum protein markers, but there are no pinCause the research of important organelle-mitochondria of oxidative damage to Indium and compounds poisoning.
ND1 genes are most conservative in chondriogen, its expression product is nadh dehydrogenase (complex I) subunit 1.The change of chondriogen content may react exogenous material have activated cellular oxidation stress process.Chondriogen contentIncrease can produce of both influence.On the one hand, it stimulates mitochondrial hyperplasia to provide energy to meet necessity of cells survivalProperty, including repair damage and synthesize new protein.On the other hand, the mitochondrial function imbalance to become increasingly abundant causes active oxygen mistakeSurplus production and further oxidative damage, possible active cell aging or death, and then cause more serious pathological consequences.
The content of the invention
It is an object of the present invention to provide the application of indium or its compound.
The present invention provides the application of indium or its compound in Mitochondrial oxidative damage is promoted.
Or,
The present invention provides indium or its compound to prepare the application in promoting Mitochondrial oxidative damage product.
In above application, the promotion Mitochondrial oxidative damage, which is embodied in, improves mitochondrial ND1 gene gene relative amount.
In above application, the mitochondrial ND1 gene gene relative amount is mitochondrial ND1 gene CT value-HGB1 genesThe value that CT is worth to.
In above application, the mitochondrial ND1 gene CT values expand to obtain by the RT-PCR of primer pair 1, the primerIt is made of to 1 the single strand dna shown in sequence 2 in the single strand dna shown in sequence in sequence table 1 and sequence table；
The Mitochondrial H GB1 gene C T values expand to obtain by the RT-PCR of primer pair 2, and the primer pair 2 is by sequence tableSingle strand dna composition in single strand dna and sequence table shown in middle sequence 3 shown in sequence 4.
In above application, the mitochondria derives from isolated cells, is specially isolated blood cell.
It is a further object to provide another of indium or its compound purposes.
The present invention provides the application of indium or its compound in mitochondrial ND1 gene gene relative amount is improved；
Or,
Indium or its compound are preparing the application in improving mitochondrial ND1 gene gene relative amount product.
In above application, the mitochondrial ND1 gene gene relative amount is mitochondrial ND1 gene CT value-HGB1 genesThe value that CT is worth to.
In above application, the mitochondrial ND1 gene CT values expand to obtain by the RT-PCR of primer pair 1, the primerIt is made of to 1 the single strand dna shown in sequence 2 in the single strand dna shown in sequence in sequence table 1 and sequence table；
The Mitochondrial H GB1 gene C T values expand to obtain by the RT-PCR of primer pair 2, and the primer pair 2 is by sequence tableSingle strand dna composition in single strand dna and sequence table shown in middle sequence 3 shown in sequence 4.
In above application, the mitochondria derives from isolated cells, is specially isolated blood cell.
In above application, the indium compound is inidum chloride.
Detect mitochondria whether the material of oxidative damage prepare diagnosis or auxiliary diagnosis body whether indium exposure or indium inApplication in the product of poison is also the scope of protection of the invention；
Or,
Whether indium is sudden and violent preparing diagnosis or auxiliary diagnosis body for the material that whether improves of ND1 gene contents in detection mitochondriaApplication in dew or the product of indium poisoning is also the scope of protection of the invention.
In above application, above-mentioned substance includes indium or its compound, and above-mentioned substance further includes primer pair 1 and primer pair 2.
The experiment proves that the present invention is cultivated by adding inidum chloride in extracorporeal blood, its blood cell lineThe change of plastochondria has differences with normal cell, it is found that inidum chloride can improve cell Mitochondria ND1 gene relative amounts.SayBright to expose process Mitochondria there occurs oxidative damage in indium, mitochondria, which quantitatively detects, can be used as Indium and compounds exposure harmPotential source biomolecule marker.
Brief description of the drawings
Fig. 1 is fluorescent PCR amplification curve diagram.
Fig. 2 is solubility curve figure.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, is commercially available unless otherwise specified (yes).
Instrument equipment is as follows in following embodiments：American AB I7300 fluorescent PCRs amplification instrument, adjustable quantitative liquid feeder,Alcolhol burner, autoclave sterilization apparatus, superclean bench, assay balance and common drug balance, blake bottle, graduated centrifuge tube (5ml,10ml), heparin sodium anticoagulant tube, disposable plastic tube, water isolation type constant incubator (outfit temperature indicating controller (TIC)), electric heating are permanentWarm drying box, electric centrifuge (horizontal)
Main matched reagent used is as follows in following embodiments：DNA extracts reagents are purchased from Beijing day bounties genome company dayNet husky serial pillar blood DNA extracts reagent, Fluorescence PCR reagent are anti-purchased from Dalian treasured biotech firm RR820A fluorescent PCRsReagent is answered, other biochemical reagents and primed probe synthetic are purchased from Shanghai Sheng Gong bio-engineering corporations.Modified form RPMI-1640 is trainedSupport base, hyclone, PHA (phytohemagglutinin), distilled water, injection heparin sodium.
The cleaning of glass tube ware and disinfection in following embodiments：Vessel are washed with washing powder first, tap water rinsesDry, then put in cleaning solution and soak 1-2 days, soaking vessel are rushed 15-20 minutes after taking out with tap water, are then used certainlyCarry out waterside trimming and shake to wash 5 times, then rushed 3 times with distilled water, at 100-120 DEG C it is dry 2 it is small when.
The autoclave sterilization of vessel in following embodiments：Prepare vessel used in culture medium it is cleaned, it is dry after, with infantees bagMake autoclaving sterilization (15 pounds 20 minutes) well, taking-up is dried for standby.
The application of embodiment 1, indium in chondriogen ND1 gene relative amounts are improved
1st, cell culture medium is prepared
Basal medium configures in accordance with the following steps：By PHA (phytohemagglutin phytolectin), heparin sodium, gentamicin sulphate, paddy amineAcid amides and RPMI-1640 nutrient solutions mix, and obtain basal medium, wherein, final concentration of 160 μ g/mL of PHA, heparin sodiumFinal concentration of 37.5IU/ml, the final concentration of 112IU/ml of gentamicin sulphate, final concentration of the 0.03% of glutamine.
Growth medium configures in accordance with the following steps：Newborn calf serum, hyclone and basal medium are mixed, obtainedTo growth medium, wherein, final concentration of 10% (volumn concentration) of calf serum, final concentration of the 10% of hyclone(volumn concentration).
2nd, blood sampling culture
15 normal, disease-free crowd (patient knows) venous blood 5ml are extracted into heparin sodium anticoagulant tube, are fully mixed, are stoodThat is censorship, obtains 15 anticoagulation samples.
3rd, cell culture
1500ul is drawn in the clean bench each sample of continuous and quantitative pipettor, each sample is divided into three groups：
0mmol/L inidum chlorides group (Normal group)：500ul is placed in blake bottle, adds 3.0ml growth mediums；
0.2mmol/L inidum chlorides group (0.2mmol/L inidum chloride exposed cell cultures group)：500ul is placed in blake bottleIn, 3.0ml growth mediums are added, and add inidum chloride to final concentration of 0.2mmol/L；
0.8mmol/L inidum chlorides group (0.8mmol/L inidum chloride exposed cell cultures group)：500ul is placed in blake bottleIn, 3.0ml growth mediums are added, and add inidum chloride to final concentration of 0.8mmol/L；
Above-mentioned three groups of blake bottles are put to 37.5 ± 0.5 DEG C of CO2Culture 3 days in incubator.
4th, chondriogen relative amount is detected
1) extraction of cell DNA
100ul cell culture fluids were drawn from three groups of blake bottles at the 3rd day of culture respectively to be used to extract cell DNA.
DNA extracts reagents are specifically carried purchased from the net husky serial pillar blood DNA extracts reagent of Beijing day bounties genome company dayTake method as follows：
The cell culture fluid that 100uL is mixed is added in 1.5ml centrifuge tubes, in the centrifuge tube of 100uL cell culture fluids0.5mL solution As (solution A has a small amount of crystal settling, uniform with before needing to rock) are added, make precipitation fully outstanding with liquid-transfering gun piping and drumingFloat, this step purpose is cell lysis, released dna, so piping and druming is more abundant better.Concussion, which mixes, treats that solution becomes clear for 30 minutesAfter bright, liquid is transferred in centrifugal adsorbing column and stands 2-5 minutes, so that DNA is fully combined with film.12,000rpm is centrifugedHalf a minute, DNA will be adsorbed onto on film, abandon the waste liquid in collecting pipe.Add the general of 0.5mL and wash column liquid, 12,000rpm centrifugations halfMinute, abandon the waste liquid in collecting pipe.Add the general of 0.5mL and wash column liquid, 12,000rpm centrifugation half a minute, abandon in collecting pipeWaste liquid.12,000rpm centrifugation half a minute, dry residual liquid.This step is critically important, with go in membrane removal to remain it is general wash column liquid, it is noSubsequent reactions can then be influenced.Centrifugal adsorbing column is placed in a new 1.5mL plastic centrifuge tubes (providing for oneself), adds appropriate 150uLGeneral eluent, room temperature are placed 2 minutes.General eluent is reused after 65 DEG C of preheatings, the effect of eluted dna can be more preferable.12,000rpm centrifugation half a minute, centrifuge tube bottom solution, that is, DNA.Can immediately using or put refrigerator and preserve for a long time.
2) RT-PCR is expanded
With the HGB1 gene primers pair shown in table 1 and ND1 gene primers to being carried out respectively to the genomic DNA of said extractedRT-PCR is expanded.
1 detection primer sequence of table
RT-PCR reaction systems use Dalian treasured biotech firm RR820A fluorescence RT PCR reaction reagents, 20 μ l of cumulative volume,Specially：Premix Ex TaqII (Tli RNaseH Plus) (2 ×) 10.0 μ l, 0.8 μ l of sense primer (10 μM),Anti-sense primer (10 μM) 0.8 μ l, ROX Reference Dye (50 ×) 0.4 μ l, DNA profiling 2.0 μ, dH2O (sterile purified water)6.0μl。
Annealing temperature is by the 1 DEG C of difference assay optimization in 55-60 DEG C of interval, and when annealing temperature is 56 degree, response curve is in typical caseS types, fluorescence signal is most strong, and 56 degree of this experimental selection carries out following all Fluorescence PCRs as annealing temperature, specificallyRT-PCR reaction conditions：95℃10s；95 DEG C of 5s, 56 DEG C of 34s, 40Cycles.
3) detect
The program making amplification curve diagram carried using PCR instrument, after reaction, draws solubility curve immediately.
Multigroup mean compares using variance analysis test, P<0.05 has significant difference.All data statistic analysis makeWith SPSS13.0 software analysis.
Quantitative PCR reacts, and the program making amplification curve diagram (Fig. 1) carried using PCR instrument, after reaction, is painted immediatelySolubility curve (Fig. 2) processed.
As PCR system at each measures absorbance after circulation terminates, just obtain using period as abscissa, inhaledShading value is the coordinate curve of ordinate, and the cycle-index corresponding to flex point of the detection sample from baseline to exponential increase is CtValue, chondriogen relative amount is ND1 gene C T value-HGB1 gene C T values.
The results are shown in Table 2.
Table 2 is fluorescent quantitative PCR CT values
AOVA variance analyses, three each group chondriogen gene relative amount mean total difference of statistical analysis.
0.8umol/L inidum chloride exposed cell cultures group, 0.2umol/L inidum chlorides exposed cell culture group and normalNo exposed cell culture group chondriogen gene relative amount be respectively quantitatively -4.49 ± 0.39, -5.02 ± 0.51 and -4.99±0.43.The mean highest of 3rd day CT value difference value wherein after the inoculation of 0.8mmol/L inidum chlorides exposed cell culture group,Higher than Normal group and 0.2mmol/L inidum chloride exposed cell culture groups.
TTEST statistics shows 0.8mmol/ inidum chlorides exposed cell culture group and 0mmol/L inidum chloride Normal groupsThe mean of CT value difference values compares, and is specifically shown in Table 3.
Inidum chloride and the comparison of infection time Peripheral Blood cell mitochondrial amplification of the table 3 for various concentrations
As can be seen that 0.8mmol/L inidum chloride exposed cell cultures group is compared with the control group, P ＜ 0.005.0.8mmol/L inidum chlorides exposed cell culture group 2P ＜ compared with 0.2mmol/L inidum chloride exposed cell culture groups0.005, it can be seen that for 0.8mmol/L groups compared with other groups, chondriogen ND1 gene relative amount differences have statistics to anticipateJustice (P<0.005), show, mitochondria is damaged.