CN107760774A - Purposes of the ABCG1 genes as the molecular marked compound of heavy ion radiation exposure diagnosis - Google Patents

Purposes of the ABCG1 genes as the molecular marked compound of heavy ion radiation exposure diagnosis Download PDF

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CN107760774A
CN107760774A CN201610700799.4A CN201610700799A CN107760774A CN 107760774 A CN107760774 A CN 107760774A CN 201610700799 A CN201610700799 A CN 201610700799A CN 107760774 A CN107760774 A CN 107760774A
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reagent
detection
heavy ion
abcg1
genes
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CN107760774B (en
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张睿凤
党旭红
杨彪
董娟聪
张忠新
王超
原雅艺
任越
刘红艳
左雅慧
段志凯
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China Institute for Radiation Protection
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China Institute for Radiation Protection
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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
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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

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Abstract

The invention belongs to the application field of gene molecule marker thing, is related to ABCG1 genes and is being prepared as molecular marked compound for the purposes in the reagent of heavy ion radiation exposure diagnosis.It is used for heavy ion radiation exposure detection using described ABCG1 genes as molecular marked compound, can be more convenient, accurate, and can determine that dose of radiation.

Description

Purposes of the ABCG1 genes as the molecular marked compound of heavy ion radiation exposure diagnosis
Technical field
The present invention generally relates to purposes of the molecular marked compound in preparing for the reagent of disease or health status diagnosis, Prepared in particular to molecular marked compound for the purposes in reagent/reagent set of radioactive exposure diagnosis.
Background technology
Ionising radiation refers to the radiation that material can be caused to ionize.Heavy ion is a kind of high linear energy transfer of dense form Ionising radiation, more serious damage can be caused to human body.In cellular level, heavy ion radiation damage mainly includes cell Survival rate decline, cell-cycle arrest, chromosome aberration etc..Damaged for heavy ion radiation to caused by human body, existing inspection Survey method mainly includes MTT colorimetric methods, flow cytometry and chromosome, micronucleus detection method, but is only the shortcomings that these methods From the degree of cell-based assay radiation injury, the molecule mechanism and micro-variations of radiation injury can not be studied.
Diagnosis/detection of heavy ion radiation is carried out using the principle selection molecular marker of molecular change after heavy ion radiation It is a kind of diagnostic method for the promising heavy ion radiation that can be largely overcoming drawbacks described above, but selects specific Detection molecules (molecular marker) need to pay close attention to following two problems：First, sensitiveness, the specificity of detection molecules change, induction The dosage that detection molecules change needs is small；Second, molecular damage caused by most of heavy ion radiations repairs fast, molecular change Have to reflect whole exposure dose of radiation in time.It is therefore more difficult to select the molecular marker evaluation for all meeting above-mentioned condition Previous radiation exposure or the degree of impairment of low-dose rate irradiation irradiation.
Biochip technology is built upon the Protocols in Molecular Biology in hybridization sequences basic theories, it with it is a kind of comprehensively, Comprehensive and system mode of thinking research biological phenomena, whole cell or organ full gene change can be intactly studied, can It is a kind of new molecular radiobiology method to find the gene to heavy ion radiation response difference by genetic analysis.Base Efficient, quick, low cost detection and analysis while can be carried out to biomolecule such as substantial amounts of nucleic acid and protein because chip has Advantage.
Albumen by ABCG1 gene codes is a member in ATP-binding boxes (ABC) transport protein superfamily, ABCG1 The various molecules of albumen Transshipment Permitted pass through intercellular membrane and outer membrane.ABC albumen be divided into seven different subfamilies (ABC1, MDR/TAP, MRP, ALD, OABP, GCN20, White), ABCG1 protein is a member of White subfamilies, participates in Macrophage cholesterol and phosphorus Fat transports, and may adjust cytolipin in other cell homeostasis.
The content of the invention
The primary and foremost purpose of the present invention is the demand for heavy ion radiation exposure diagnosis, there is provided molecular marked compound is used in preparation Purposes in reagent/reagent set of heavy ion radiation exposure diagnosis, with more convenient, accurate, and can determine that dose of radiation.
In order to achieve this, in the embodiment on basis, the present invention provides ABCG1 genes and made as molecular marked compound The purposes being ready for use in reagent/reagent set of heavy ion radiation exposure diagnosis.
In a preferred embodiment, the present invention provides ABCG1 genes and prepared as molecular marked compound for weight Purposes in reagent/reagent set of ionizing radiation exposure diagnosis, wherein described heavy ion is12C6+Ion.
In a preferred embodiment, the present invention provides ABCG1 genes and prepared as molecular marked compound for weight Purposes in reagent/reagent set of ionizing radiation exposure diagnosis, wherein heavy ion radiation dosage is 0.1-2.0Gy.
In a preferred embodiment, the present invention provides ABCG1 genes and prepared as molecular marked compound for weight Purposes in reagent/reagent set of ionizing radiation exposure diagnosis, wherein described reagent/reagent set includes being used for PCR detections, base Because the whole of chip detection, the detection of southern blot hybridizations, the detection of northern blot hybridizations or in situ hybridization detection is required Reagent.
In a preferred embodiment, the present invention provides ABCG1 genes and prepared as molecular marked compound for weight Purposes in reagent/reagent set of ionizing radiation exposure diagnosis, wherein described reagent/reagent set includes being used for PCR detections All must reagent.
In a kind of preferred embodiment, the present invention provide ABCG1 genes as molecular marked compound prepare be used for it is heavy from Purposes in reagent/reagent set of sub- radioactive exposure diagnosis, wherein for PCR detections all must reagent include following sequence Primer：
F：5 '-ATCATTTGCACCATCCACCAG-3 ',
R:5’-TGGTAGGTTGGGCAGTTCAGAC-3’。
It is a further object to provide reagent/reagent set for heavy ion radiation exposure diagnosis, so that diagnosis It is more convenient, accurate, and can determine that dose of radiation.
In order to achieve this, in the embodiment on basis, the present invention is provided to the examination of heavy ion radiation exposure diagnosis Agent/reagent set, it include for ABCG1 genetic tests all must reagent.
In a preferred embodiment, the present invention is provided to heavy ion radiation to expose the reagent/reagent set diagnosed, Wherein described reagent/reagent set include for PCR detections, genechip detection, southern blot hybridizations detect, What the detection of northern blot hybridizations or in situ hybridization detected all must reagent.
In a preferred embodiment, the present invention is provided to heavy ion radiation to expose the reagent/reagent set diagnosed, Wherein described reagent/reagent set include be used for PCR detection all must reagent.
In a preferred embodiment, the present invention is provided to heavy ion radiation to expose the reagent/reagent set diagnosed, Wherein be used for PCR detections all must reagent include the primer of following sequence：
F：5 '-ATCATTTGCACCATCCACCAG-3 ',
R:5’-TGGTAGGTTGGGCAGTTCAGAC-3’。
Embodiment
The embodiment of the present invention is further illustrated with reference to embodiments.
Embodiment 1:Preliminary screening for the gene molecule mark of heavy ion radiation exposure diagnosis
Using biochip technology, by the difference table for detecting different genes in human lymphocyte after heavy ion radiation irradiation Reach, preliminary screening is used for the gene molecule mark of heavy ion radiation exposure diagnosis.
1) the extracting, quantify of cell total rna, quality inspection and purifying
By human lymphocyte strain through 0.1,0.5,2.0Gy12C6+24h is cultivated after ionizing radiation irradiation, and (this three groups are experiment Group；Non- radiation exposure is control group).Four groups of cells are according to Trizol reagents extraction specification extraction total serum IgE.Spectrophotometric measures Concentration and purity, total serum IgE 5g electrophoresis in 1% formaldehyde-agarose gel is taken to examine.According to QIAGENKit (QIAGEN companies, Germany) specification purifying total serum IgE.
2) cDNA is synthesized
0.2 μ g RNA are taken to configure following reaction solution in 0.2ml centrifuge tubes：200ng total serum IgEs (5ng polyadenylic acids The μ l of polyA+RNA 2.5, μ l of 2 μ l, T7 primer of dilution 0.8), 65 DEG C of insulations 10min, ice bath 5min；Configure cDNA compound bodies System：μ l, the 10mM dezyribonucleosides of 5 × the first 2 μ l, 0.1M dithiothreitol (DTT)s of chain mixed liquor First Strand Buffer 1 The sour μ l of 0.5 μ l, AffinityScript Rnase Block mixed liquors of mixed liquor 1.2, by above-mentioned 4.7 μ l cDNA synthetic systems After addition denaturation in the RNA of ice bath, centrifuged after mixing；According to 40 DEG C of 2h, 70 DEG C of 15min, ice bath 5min PCR response procedures close Into cDNA.
3) fluorescence labeling cRNA, cRNA purifying and Quality Control
Add 6 μ l transcription mixed liquors (H2The μ l of O 0.75, the μ l cores of 5 × transcription buffer, 3.2 μ l, 0.1M dithiothreitol (DTT) 0.6 μ l of 1 μ l, Cy3- atriphos of ribotide mixed liquor, 0.21 μ l, T7 RNA polymerases mixed liquor 0.24) mix, 40 DEG C of placements 2h；According to QIAGEN RNeasy Mini kit kits (QIAGEN companies, Germany) operation manual purifying cRNA；Spectrophotometric Meter analysis RNA concentration.
4) cRNA sample fragmentizations and chip hybridization, washing, scanning
Upper chip, 65 DEG C of 17h, 10rpm after cRNA sample fragments are rolled into hybridization；After taking-up respectively in washing lotion 1,2 Washing 1 minute；Scanned in Agilent scanners, resolution ratio is 5 μm, and scanner is automatically with 100% and 10%PMT respectively scannings one Secondary, two times result Agilent softwares can merge automatically.
Fold differences (Fold change, the difference of obtained significance of difference P values and normalized signal value are examined using T Multiple=log2(experimental group scanning signal value/control group scanning signal value)) to be screened, standard is fold differences value>=2.0 And P values<=0.05；On the occasion of representing to raise, negative value represents to lower fold differences value.
Difference expression gene is carried out to analyze visible, the fold differences of the portion gene of three common differential expressions of dosage group Dose-dependence be present with exposure dose of radiation, part variation multiple up-regulated gene is listed in table 1, and wherein ABCG1 genes are with agent Amount increase, fold differences gradually rise, and the fold differences significant difference of various dose, therefore choose ABCG1 genes and enter to advance One_step PCR is verified.
The co expression up-regulated gene of dosage effect be present in table 1
Embodiment 2：PCR checking confirm ABCG1 genes as molecular marked compound be used for heavy ion radiation exposure diagnose can Row
Radiation exposure group and non-radiation exposure group cell total rna, ultraviolet specrophotometer measure are extracted by Trizol reagents Concentration, using total serum IgE as template, concrete operation step presses Reverse Transcriptase kit specification reverse transcription cDNA, and reverse transcription system is 20 μ l。
Appropriate radiation exposure group and control group sample cDNA are taken, performing PCR checking is entered to ABCG1 difference expression genes.It draws Thing sequence and PCR amplification conditions are shown in Table 2.
The primer sequence and amplification condition of the RT-PCR amplification genes of table 2
Interpretation of result：For three dosage groups compared with cellular control unit, common difference expression gene has 504, wherein on Adjust gene 88, down-regulated gene 416.And it was found that the fold differences of part variation expressing gene exist with exposure dose of radiation The fold differences up-regulation of dose response relation, wherein various dose ABCG1 genes is notable.Further PCR checkings hair is carried out to it It is existing：The result is consistent with gene chip results (being shown in Table 3).
The relative quantification result of the RT-PCR of table 3 amplifications
Note:*P<0.05 radiation exposure group has statistical significance compared with control group；
F=2-△△ct, wherein：
△ △ ct=△ ct (to be measured group)-△ ct (control group)
△ ct (to be measured group)=average ct values of the average ct values-to be measured group house-keeping gene of to be measured group of target gene
The average ct values of the average ct values-control group house-keeping gene of △ ct (control group)=control group target gene.
From table, warp12C6+The expression of ABCG1 genes raises compared with control group after ionizing radiation irradiation, and with Dosage increase, fold differences increase.Therefore the molecular marked compound that is exposed as heavy ion radiation of screening ABCG1 genes, as by According to an index of personnel health's monitoring.
Above-described embodiment is for example, the present invention can also be with other ad hoc fashions or others to the present invention Particular form is implemented, without departing from idea of the invention or substantive characteristics.Therefore, from the point of view of the embodiment of description is in terms of any It is regarded as illustrative and non-limiting.The scope of the present invention should illustrate by appended claims, any and claim Intention and the equivalent change of scope also should be within the scope of the present invention.

Claims (10)

1.ABCG1 genes are being prepared for the use in reagent/reagent set of heavy ion radiation exposure diagnosis as molecular marked compound On the way.
2. purposes according to claim 1, it is characterised in that described heavy ion is12C6+Ion.
3. purposes according to claim 1, it is characterised in that heavy ion radiation dosage is 0.1-2.0Gy.
4. purposes according to claim 1, it is characterised in that described reagent/reagent set includes being used for PCR detections, gene Chip detection, the detection of southern blot hybridizations, the detection of northern blot hybridizations or in situ hybridization detection must all try Agent.
5. purposes according to claim 1, it is characterised in that described reagent/reagent set includes being used for the complete of PCR detections The required reagent in portion.
6. purposes according to claim 5, it is characterised in that for PCR detection all must reagent include following sequence Primer：
F：5 '-ATCATTTGCACCATCCACCAG-3 ',
R:5’-TGGTAGGTTGGGCAGTTCAGAC-3’。
7. reagent/reagent set for heavy ion radiation exposure diagnosis, it is characterised in that including for the complete of ABCG1 genetic tests The required reagent in portion.
8. reagent/reagent set according to claim 7, it is characterised in that described reagent/reagent set includes being used for PCR inspections The whole that survey, genechip detection, the detection of southern blot hybridizations, the detection of northern blot hybridizations or in situ hybridization detect Required reagent.
9. reagent/reagent set according to claim 7, it is characterised in that described reagent/reagent set includes being used for PCR inspections Survey all must reagent.
10. reagent/reagent set according to claim 9, it is characterised in that for PCR detection all must reagent include The primer of following sequence：
F：5 '-ATCATTTGCACCATCCACCAG-3 ',
R:5’-TGGTAGGTTGGGCAGTTCAGAC-3’。
CN201610700799.4A 2016-08-22 2016-08-22 Application of ABCG1 gene as molecular marker for heavy ion radiation exposure diagnosis Active CN107760774B (en)

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Cited By (1)

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CN110218767A (en) * 2019-06-24 2019-09-10 南华大学 A method of low dosage gamma ray radiation, which is evaluated, using glutathione peroxidase 4 damages

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Publication number Priority date Publication date Assignee Title
CN110218767A (en) * 2019-06-24 2019-09-10 南华大学 A method of low dosage gamma ray radiation, which is evaluated, using glutathione peroxidase 4 damages
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