CN107760775B - Use of APOL6 gene as molecular marker for heavy ion radiation exposure diagnosis - Google Patents

Use of APOL6 gene as molecular marker for heavy ion radiation exposure diagnosis Download PDF

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CN107760775B
CN107760775B CN201610700864.3A CN201610700864A CN107760775B CN 107760775 B CN107760775 B CN 107760775B CN 201610700864 A CN201610700864 A CN 201610700864A CN 107760775 B CN107760775 B CN 107760775B
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apol6
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张睿凤
董娟聪
林海鹏
党旭红
张忠新
王超
原雅艺
任越
刘建功
左雅慧
段志凯
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China Institute for Radiation Protection
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Abstract

The invention belongs to the application field of gene molecular markers, and relates to application of an APOL6 gene as a molecular marker in preparation of a reagent for heavy ion radiation exposure diagnosis. The APOL6 gene is used as a molecular marker for heavy ion radiation exposure detection, so that the detection is more convenient and accurate, and the radiation dose can be determined.

Description

Use of APOL6 gene as molecular marker for heavy ion radiation exposure diagnosis
Technical Field
The present invention relates generally to the use of molecular markers for the preparation of reagents for the diagnosis of diseases or health conditions, and in particular to the use of molecular markers for the preparation of reagents/reagent sets for the diagnosis of radiation exposure.
Background
Ionizing radiation refers to radiation that can cause ionization of a substance. Heavy ions are dense ionizing radiation with high energy transmission linear density, and can cause serious damage to human bodies. At the cellular level, heavy ion radiation damage mainly includes decreased survival of cells, cell cycle arrest, chromosomal aberrations, and the like. Aiming at the damage to a human body caused by heavy ion radiation, the existing detection methods mainly comprise an MTT colorimetric method, flow cytometry and chromosome and micronucleus detection methods, but the methods have the defects that the degree of radiation damage can be detected only from a cellular level, and the molecular mechanism and microscopic change of the radiation damage cannot be observed.
The diagnosis/detection of heavy ion radiation by selecting molecular markers based on the principle of molecular change after heavy ion radiation is a promising diagnosis method of heavy ion radiation that can overcome the above-mentioned drawbacks to a great extent, but the selection of specific detection molecules (molecular markers) requires attention to the following two problems: firstly, the sensitivity and the specificity of detecting molecular change are realized, and the dosage required for inducing the detection molecular change is small; secondly, most of the molecular damage caused by heavy ion radiation is repaired quickly, and the molecular change must reflect the whole radiation dose in time. Therefore, it is difficult to select all molecular markers satisfying the above conditions to evaluate the damage caused by the previous irradiation or the low dose rate irradiation.
The gene chip technology is a molecular biology technology established on the basic theory of hybrid sequences, which researches life phenomena in a comprehensive, comprehensive and systematic way, can completely research all gene changes of whole cells or organs, can discover genes with different responses to heavy ion radiation through gene analysis, and is a novel molecular radiobiology method. The gene chip has the advantages of high efficiency, rapidness and low cost detection and analysis on a large number of biomolecules such as nucleic acid, protein and the like.
The APOL6 gene, apolipoprotein L gene family, belongs to the programmed cell death regulatory gene family, and its protein can induce apoptosis or autophagic death of a subject. In humans, the apolipoprotein L gene family consists of six genes, located on human chromosome 22.
Disclosure of Invention
The primary object of the present invention is to provide the use of molecular markers for preparing reagents/reagent sets for heavy ion radiation exposure diagnosis, aiming at the requirements of heavy ion radiation exposure diagnosis, so as to be more convenient, accurate and capable of determining radiation dosage.
To achieve this, in a basic embodiment, the present invention provides the use of the APOL6 gene as a molecular marker for the preparation of a reagent/reagent set for diagnosis of heavy ionizing radiation exposure.
In a preferred embodiment, the present invention provides the use of the APOL6 gene as a molecular marker for the preparation of a reagent/kit for the diagnosis of heavy ion radiation exposure wherein the heavy ion is12C6+Ions.
In a preferred embodiment, the present invention provides the use of the APOL6 gene as a molecular marker for the manufacture of a reagent/reagent set for diagnosis of heavy ionizing radiation exposure wherein the dose of heavy ionizing radiation is 0.1-2.0 Gy.
In a preferred embodiment, the present invention provides the use of the APOL6 gene as a molecular marker for the preparation of a reagent/reagent set for heavy ion radiation exposure diagnosis, wherein said reagent/reagent set comprises all the necessary reagents for PCR detection, gene chip detection, southern blot hybridization detection, northern blot hybridization detection or in situ hybridization detection.
In a preferred embodiment, the present invention provides the use of the APOL6 gene as a molecular marker in the preparation of a reagent/set of reagents for heavy ionizing radiation exposure diagnostics, wherein said reagent/set of reagents comprises all the necessary reagents for PCR detection.
In a preferred embodiment, the present invention provides the use of the APOL6 gene as a molecular marker in the preparation of a reagent/reagent set for heavy ionizing radiation exposure diagnosis, wherein all the necessary reagents for PCR detection include primers of the following sequences:
F：5’-GCAAGGACAGAGGTTCAGGAT-3’，
R：5’-AGCCTCTGTGGCAGCAAAT-3’。
it is another object of the present invention to provide a reagent/reagent set for heavy ionizing radiation exposure diagnosis, so that diagnosis is more convenient, accurate, and radiation dosage can be determined.
To achieve this, in a basic embodiment, the present invention provides a reagent/reagent set for heavy ionizing radiation exposure diagnosis, which includes all the necessary reagents for the detection of the APOL6 gene.
In a preferred embodiment, the present invention provides a reagent/reagent set for heavy ion radiation exposure diagnosis, wherein the reagent/reagent set comprises all necessary reagents for PCR detection, gene chip detection, southern blot hybridization detection, northern blot hybridization detection or in situ hybridization detection.
In a preferred embodiment, the present invention provides a reagent/reagent set for heavy ionizing radiation exposure diagnosis, wherein said reagent/reagent set comprises all necessary reagents for PCR detection.
In a preferred embodiment, the present invention provides a reagent/reagent set for heavy ionizing radiation exposure diagnosis, wherein all necessary reagents for PCR detection include primers of the following sequences:
F：5’-GCAAGGACAGAGGTTCAGGAT-3’，
R：5’-AGCCTCTGTGGCAGCAAAT-3’。
Detailed Description
The following examples further illustrate embodiments of the present invention.
Example 1 preliminary screening of Gene molecular markers for diagnosis of heavy ion radiation Exposure
By using gene chip technology, different gene expression in human lymphocyte is detected after irradiation of heavy ion radiation, and gene molecular marker for heavy ion radiation exposure diagnosis is primarily screened.
1) Extraction, quantification, quality inspection and purification of total RNA of cells
Subjecting human lymphocyte cell strain to 0.1, 0.5, 2.0Gy12C6+After irradiation with ionizing radiation, the cells were incubated for 24 hours (these three groups were experimental groups; non-irradiated control group). Four groups of cells were extracted for total RNA according to Trizol reagent extraction instructions. Measuring concentration and purity with spectrophotometer, collecting total RNA 5g in 1% formaldehyde-agarose gelAnd (4) performing electrophoresis inspection. According to QIAGEN
Figure BDA0001085983490000031
Kit (QIAGEN, Germany) instructions purify total RNA.
2) cDNA Synthesis
Take 0.2. mu.g RNA in 0.2ml centrifuge tube, prepare the following reaction solution: 200ng of total RNA (5ng poly A + RNA 2.5. mu.l, 2. mu.l of diluent, 0.8. mu.l of T7 primer), heat preservation at 65 ℃ for 10min, ice bath for 5 min; preparing a cDNA synthesis system: 5 multiplied first strand mixed solution FirstStrand Buffer 2 mul, 0.1M dithiothreitol 1 mul, 10mM deoxyribonucleotide mixed solution 0.5 mul, AffinityScript RNase Block mixed solution 1.2 mul, adding the 4.7 mul cDNA synthesis system into the RNA after denaturation and ice bath, mixing evenly and centrifuging; the cDNA was synthesized according to the PCR reaction procedure at 40 ℃ for 2h, 70 ℃ for 15min, and ice bath for 5 min.
3) Fluorescence labeling cRNA, cRNA purification and quality control
Add 6. mu.l of transcription mix (H)2O0.75. mu.l, 5 Xtranscription buffer solution 3.2. mu.l, 0.1M dithiothreitol 0.6. mu.l ribonucleotide mixture 1. mu.l, Cy 3-adenosine triphosphate 0.21. mu.l, T7RNA polymerase mixture 0.24. mu.l) and standing at 40 ℃ for 2 hours; the cRNA was purified according to the QIAGEN RNeasy Mini kit (QIAGEN, germany) operating manual; RNA concentration was analyzed spectrophotometrically.
4) Fragmentation of cRNA sample, hybridization of chip, washing and scanning
Fragmenting a cRNA sample, loading the chip, and carrying out rolling hybridization at 65 ℃ for 17h and 10 rpm; taking out and washing in washing liquid 1 and 2 for 1 minute; scanning in an Agilent scanner with the resolution of 5 μm, automatically scanning once by the scanner with 100% and 10% PMT respectively, and automatically combining the two results by Agilent software.
Fold difference between significance P-value and normalized signal value by T-test (Foldchange, log ═ difference2(experimental group scan signal value/control group scan signal value)) were screened using a standard of fold difference value>2.0 and P value<0.05; fold difference values are positive for up-regulation and negative for down-regulation.
Analysis on the differentially expressed genes shows that dose-dependent relationship exists between the fold difference of partial genes differentially expressed by the three dose groups and the radiation irradiation dose, and the partial fold difference up-regulated genes are listed in table 1, wherein the fold difference of the APOL6 gene gradually decreases with the increase of the dose, and the difference of the fold difference of different doses is obvious, so that the APOL6 gene is selected for further PCR verification.
TABLE 1 Co-expression of Up-regulated genes with dose Effect
Figure BDA0001085983490000041
Figure BDA0001085983490000051
Example 2: PCR verification for confirming feasibility of APOL6 gene as molecular marker for heavy ion radiation exposure diagnosis
Extracting total RNA of cells of a radiation irradiation group and a non-radiation irradiation group according to Trizol reagent, measuring the concentration by an ultraviolet spectrophotometer, and carrying out reverse transcription on cDNA according to the specification of a reverse transcription kit by taking the total RNA as a template, wherein the reverse transcription system is 20 mu l.
And taking a proper amount of sample cDNA of the radiation irradiation group and the control group, and carrying out PCR verification on the APOL6 differential expression gene. The primer sequences and PCR amplification conditions are shown in Table 2.
TABLE 2 primer sequences and amplification conditions for RT-PCR amplified genes
Figure BDA0001085983490000061
And (4) analyzing results: the three dose groups had 504 genes differentially expressed in common compared to the control cells, 88 genes were up-regulated and 416 genes were down-regulated. And the dose response relationship of the difference multiple of partial differential expression genes and the radiation irradiation dose is found, wherein the difference of the difference multiple of APOL6 genes in different doses is obvious. Further PCR verification of the DNA sequence shows that: the results of the verification were consistent with those of the gene chip (see Table 3).
TABLE 3 relative quantification results of RT-PCR amplification
Figure BDA0001085983490000062
Compared with a control group, the P <0.05 radiation irradiation group has statistical significance;
F＝2-ΔΔctwherein:
Δ Δ ct ═ Δ ct (test group) - Δ ct (control group)
(delta ct (test group) ═ test group target gene average ct value-test group housekeeping gene average ct value
Δ ct (control group) is the average ct value of the target gene of the control group-the average ct value of the housekeeping gene of the control group.
As can be seen from the table, the channels12C6+The level of expression of the APOL6 gene was up-regulated after ionizing radiation exposure compared to the control group and the fold difference decreased with increasing dose. Therefore, the APOL6 gene is screened as a molecular marker of heavy ion radiation exposure as an index of health monitoring of the irradiated personnel.
The above-described embodiments are merely illustrative of the present invention, which may be embodied in other specific forms or in other specific forms without departing from the spirit or essential characteristics thereof. The described embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. The scope of the invention should be indicated by the appended claims, and any changes that are equivalent to the intent and scope of the claims should be construed to be included therein.

Claims (1)

  1. Use of the APOL6 gene as a molecular marker for the preparation of a reagent/set of reagents for the diagnosis of heavy ion radiation exposure of human lymphocytes, wherein said heavy ion is12C6+Ion, heavy ion radiation dose is 0.1-2.0Gy, the reagent/reagent set comprises all necessary reagents for PCR detection, and all necessary reagents for PCR detection comprise primers with the following sequences:
    F：5’-GCAAGGACAGAGGTTCAGGAT-3’，
    R：5’-AGCCTCTGTGGCAGCAAAT-3’。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101044249A (en) * 2004-10-20 2007-09-26 日立化成工业株式会社 Method for tailoring administration of drugs by quantitation of mrna
WO2013082722A1 (en) * 2011-12-09 2013-06-13 British Columbia Cancer Agency Branch Predicting prognosis in classic hodgkin lymphoma
CN103820552A (en) * 2014-02-26 2014-05-28 东华大学 Real-time quantification PCR chip used for detecting gene expression of mouse cholesterol metabolism

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101044249A (en) * 2004-10-20 2007-09-26 日立化成工业株式会社 Method for tailoring administration of drugs by quantitation of mrna
WO2013082722A1 (en) * 2011-12-09 2013-06-13 British Columbia Cancer Agency Branch Predicting prognosis in classic hodgkin lymphoma
CN103820552A (en) * 2014-02-26 2014-05-28 东华大学 Real-time quantification PCR chip used for detecting gene expression of mouse cholesterol metabolism

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
《６０Ｃｏγ辐射致小鼠血液中ｍｉＲＮＡ表达改变及意义》;李刚强等;《中华实用诊断与治疗杂志》;20160430;第30卷(第4期);全文 *
《Homo sapiens apolipoprotein L6 (APOL6), mRNA》;Ye Q等;《NCBI Reference Sequence: NM_030641.4》;20010327;参见参考文献1序列及其注释 *
《重离子生物效应的研究进展》;张睿凤等;《国际放射医学和医学杂志》;20160731;第40卷(第4期);全文 *

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