CN114277122A

CN114277122A - Application of peripheral blood leukocyte miR-150-5p in calculating radiation dose

Info

Publication number: CN114277122A
Application number: CN202111633929.4A
Authority: CN
Inventors: 程赢; 刘聪; 杜继聪; 陈伟; 高福; 杨彦勇
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2021-12-29
Filing date: 2021-12-29
Publication date: 2022-04-05

Abstract

The invention provides application of peripheral blood leukocyte miR-150-5p in calculating radiation dose. The method mainly comprises the steps of detecting the expression quantity of miR-150-5p in peripheral blood leukocytes of organisms after the irradiation, substituting the expression quantity into a dose-effect function relation, and calculating the irradiation quantity. The method is simple to operate, convenient, rapid and objective.

Description

Application of peripheral blood leukocyte miR-150-5p in calculating radiation dose

Technical Field

The invention relates to the technical field of biology, in particular to application of peripheral blood leukocyte miR-150-5p in radiation dose calculation.

Background

Under the condition that nuclear reactor accidents and nuclear weapon explosions happen to nuclear power vessels, people can suffer from acute radiation diseases with different degrees after being irradiated by ionizing radiation with different doses, and the severity and the typing graduation of the acute radiation diseases are closely related to the irradiated dose. Therefore, the rapid and accurate evaluation of the irradiated dose of the personnel has important guiding significance for scientifically judging the severity of the acute radiation disease and clinically carrying out graduation and staged treatment on the acute radiation disease.

The existing ionizing radiation external irradiation dose estimation methods have three types: physical, biological and clinical, but because the physical dosimeter can only reflect local irradiated dose, and the response of the irradiated personnel to the ray has individual difference; clinical diagnosis is difficult to make a quick diagnosis of the wounded, so that estimation of the irradiated dose by biological indicators is still not an alternative. The use of biological index analysis to assess the biological dose of a person being irradiated is currently the field of greatest interest in radiobiology and radiation protection research.

Although many biological indicators with radiation dose correlation are proved by research, the practical application is still less, and the application is more, and the biological dosimeter is still at a cellular level. Chromosome aberration analysis has been the gold standard for radiation bio-dose estimation, but it is highly demanding on the analyst's personal skill and experience. And the radiation dose is estimated through the expression or mutation of a single gene, so that the randomness is high, the influence of environmental factors such as smoking is easy to realize, and the individual difference is large. There is a great need to find new methods for scientific and rapid assessment of radiation dose.

Disclosure of Invention

In view of the shortcomings of the prior art, the invention aims to provide application of peripheral blood leukocyte miR-150-5p in calculating radiation dose, which is used for solving the problems that the radiation dose is difficult to determine and the existing operation is complicated in the prior art.

To achieve the above and other related objects, another aspect of the present invention provides a method for calculating the dose of radiation to which an organism is subjected, by detecting the expression level of miR-150-5p in peripheral blood leukocytes of the organism after 36-52 h of radiation exposure, and substituting the expression level into a dose-effect function relationship:

D＝-2.864*lg(X_miR-150-5p)-0.0009797 r²＝0.9026

wherein D is the irradiation dose, and X is the relative expression quantity of miRNA, and the irradiation dose can be obtained by calculation.

The method for detecting the expression level of miRNA in peripheral blood leukocytes of an organism may be any of various detection methods known in the art, for example, a second generation sequencing detection method, or other known detection methods.

Preferably, the peripheral blood is obtained 48h after the organism has been subjected to the radiation in the method.

Another aspect of the invention provides a product for calculating a radiation dose to which an organism is subjected, the product comprising the following calculation modules: D-2.864X lg (X)_miR-150-5p)-0.0009797 r²＝0.9026

Wherein D is irradiation dose, and X is miRNA relative expression.

The product can be a microcomputer, and the function relation is embedded in the microcomputer, so that the radiation dose can be immediately calculated as long as a user inputs the relative expression quantity of each miRNA.

Preferably, the product also comprises a detection reagent for detecting the miRNA in the peripheral blood.

Preferably, the detection reagents include, without limitation: and (3) a second-generation sequencing detection reagent.

Another aspect of the present invention provides a computer storage medium having a computing module as described above incorporated therein.

As described above, the method for estimating the biological dose of radiation based on miRNA in a group of peripheral blood leukocytes according to the present invention has the following beneficial effects:

the method is convenient and quick, is convenient to operate, and can quickly estimate the radiation dose suffered by the organism.

Drawings

FIG. 1 is a graph showing the dose-response curves between the relative expression amounts of miRNA and the corresponding irradiation doses obtained by calculation in example 1

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It is to be understood that the processing equipment or apparatus not specifically identified in the following examples is conventional in the art.

Brief description of the construction process of the present technical solution:

1) selecting C57BL/6 mice to be divided into 5 groups (0, 2, 4, 6 and 8Gy) to be respectively irradiated by gamma rays, collecting peripheral blood of the mice respectively 24 hours and 48 hours after irradiation, detecting the change of miRNA expression profile in leucocytes by adopting second-generation sequencing, and screening miRNA with stable expression level and obvious change.

The mirnas we screened should meet the following criteria: a. the radiation sensitivity is high, namely the up-regulation or down-regulation change of the expression quantity before and after irradiation needs to be as large as possible; b. the dependency relationship between the expression quantity and the irradiation dose is stable, namely the miRNA expression is definitely dependent on the irradiation dose; mirnas are present in both mice and humans.

In addition, although the basal expression level of miRNA cannot be completely consistent for each mouse, this difference is small relative to its variation and therefore does not affect the judgment of the results.

Experiments show that the expression level of miR-150-5p changes remarkably before and after irradiation, and has extremely high sensitivity.

2) And combining the up-regulation or down-regulation times of the miR-150-5p expression quantity relative to the non-irradiated group with the known corresponding irradiation dose to establish a dose-effect function relation between the irradiation dose and the up-regulation or down-regulation times of the miR-150-5p corresponding expression quantity.

In specific application, the irradiated dose can be estimated by detecting the times of up-regulation or down-regulation of miR-150-5p expression quantity in peripheral blood leukocytes of mice with unknown irradiation dose and bringing the expression quantity into the previously established dose-effect function.

The inventor finally determines one miRNA (miR-150-5p) in peripheral blood leukocytes 48h after the selection of the control through experiments, and establishes a dose-effect function relation according to the expression quantity change multiple and the irradiated dose:

D＝-2.864*lg(X_miR-150-5p)-0.0009797 r²＝0.9026

wherein D is irradiation dose, and X is miRNA relative expression.

The following is a specific application of the present disclosure by way of specific examples.

Example 1 verification of the method for estimating the radiation biological dose of miRNA in peripheral blood leukocytes

1. Blood sample collection: 1ml of mouse peripheral blood with known irradiation dose is taken to be vibrated and anticoagulated in an anticoagulation tube, and centrifuged for 10 minutes at 3000rpm at 4 ℃ by a low-temperature centrifuge, and 200ul of supernatant plasma is taken; and adding 3ml of erythrocyte lysate into the cell sediment, uniformly blowing and standing for 10 minutes (shaking twice with the middle reversed), then centrifuging for 10 minutes at 4 ℃ and 3000rpm of a low-temperature centrifuge, adding 1ml of sterile PBS into the sediment, uniformly blowing and washing, then centrifuging for 10 minutes at 4 ℃ and 3000rpm of the low-temperature centrifuge to obtain leukocyte sediment, and re-suspending the leukocyte sediment to 200ul by using the PBS.

2. RNA extraction: the extraction of total RNA in the sample is carried out by adopting a MagaBio plus total RNA purification kit BSC53S1E and a matched NPA-32P nucleic acid purifier. And (3) inverting the 96-pore plate of the BSC53S1E kit for 3 times at room temperature, removing the plastic packaging film, and throwing hands for several times to avoid hanging liquid. The aluminum foil film of the 96-well plate was then torn off and the plate orientation was confirmed. Adding 200ul of ML Buffer into the white blood cells, shaking and mixing uniformly, standing for 10 minutes, transferring into a 96-well plate, putting the 96-well plate into an NPA-32P nucleic acid purification instrument, installing a magnetic bar sheath, and starting an automatic program to extract RNA.

3. Determination of RNA concentration: the concentration of the extracted total RNA was determined using a Nano Drop nucleic acid quantifier. After the RNA quantitative program is started, the upper and lower needle tip bases of the instrument are cleaned by RNase-free water, and the experimental absorbent paper is blotted. Absorbing 2.5ul RNase-free water sample for detecting background, then absorbing 2.5ul RNA sample for detecting, and recording the sample concentration.

4. Reverse transcription of miRNA: one cDNA synthesized by PolyA tailing reaction can be used for detecting multiple miRNAs simultaneously, and the following reaction mixture is added into a 0.2ml RNase-free centrifuge tube in an ice bath: (different kits are slightly different)

Reagent	Volume (ul)
		2×mRQ Buffer	5
miRNA sample	(0.25-0.8ug)
		mRQ Enzyme	1.25
RNase-free H₂o	Make up 10ul
		Total volume	10ul

The tube was gently mixed and centrifuged for 3 to 5 seconds, and then incubated at 37 ℃ for 15 minutes, then heated at 85 ℃ for 5 seconds to inactivate the enzyme, and then stored at 4 ℃. Adding 90ul dd H₂o constant volume to 100 ul. And the method can be used for subsequent quantitative PCR detection.

5. And (3) miRNA quantitative detection: the PCR reaction mixture was prepared on an ice bath with the following components:

reagent	Volume (ul)
		dd H₂o	9
2×TB Green Advantage Premix	12.5
		50×ROX Dye	0.5
miRNA-specific primer(10uM)	0.5
		mRQ 3’primer(10uM)	0.5
cDNA	2
		Total volume	25ul

Setting corresponding parameters according to the quantitative PCR instrument used

1. Pre-denaturation (1 cycle) 30 seconds at 95 ℃

2. PCR reaction (40 cycles)

95 ℃ for 5 seconds

60 ℃ for 34 seconds

3. Dissolution Curve (1 cycle)

95 ℃ for 15 seconds

60 ℃ for 60 seconds

95 ℃ for 1 second

Ct values (Cycle Threshold) were recorded and the relative expression was obtained by the following formula:

relative expression amount of 2^{- [ Ct (purpose detection-housekeeping detection) -Ct (purpose control-housekeeping control)]}

6. Calculation of dose estimates: setting 3 multiple holes for detection of each sample, finally dividing the relative expression quantity of each dosage point by the relative expression quantity of 0Gy to serve as the up-regulation or down-regulation multiple of the miRNA, substituting the up-regulation and down-regulation multiple of each miRNA in the fitting curve into an equation, and obtaining the estimated value of the irradiated dosage, wherein the specific experimental results are shown in tables 1 and 2 and figure 1, and the analysis results are shown in table 2.

TABLE 1

TABLE 2

The estimation accuracy is higher the smaller the mean square error is, and the estimation accuracy is lower the larger the mean square error is. As can be seen from Table 2, the dose-effect function relationship constructed by the method is simpler, more convenient and faster to operate and has more objective results compared with the existing method.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Claims

1. A method for calculating the radiation dose of an organism, which is to detect the expression quantity of miR-150-5p in peripheral blood leukocytes of the organism after being subjected to radiation for 36h-52h and substitute the expression quantity into a dose-effect function relation:

D＝-2.864*lg(X_miR-150-5p)-0.0009797 r²＝0.9026；

2. The method of claim 1, wherein: the method for detecting the peripheral blood miRNA expression amount in the method is a second-generation sequencing detection method.

3. The method of claim 2, wherein: the time for obtaining the peripheral blood in the method is 48 hours after the organism is subjected to the radiation.

4. A product for calculating a radiation dose to which a living being is exposed, characterized by: the product comprises the following calculation modules:

D＝-2.864*lg(X_miR-150-5p)-0.0009797 r²＝0.9026，

wherein D is irradiation dose, and X is miRNA relative expression.

5. The product of claim 4, wherein: the product also comprises a detection reagent for detecting miRNA in peripheral blood.

6. The product of claim 5, wherein: the detection reagents include, but are not limited to: and (3) a second-generation sequencing detection reagent.

7. A computer storage medium containing a computing module as claimed in claim 4.