CN109295200B - Biological detection marker for high linear energy transfer ray radiation and application thereof - Google Patents
Biological detection marker for high linear energy transfer ray radiation and application thereof Download PDFInfo
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Abstract
The invention discloses a biological detection marker for high linear energy transfer ray radiation and application thereof, and the invention analyzes and screens 12 biological detection markers Retnlb, S100a8, Csf3r, Slc4a1, Tubb1, Clec4d, Il1b, Apol11b, Alas2, Coro1a, Ahsp and Rsad2 with high LET specificity sensitivity. The 12 specific gene combinations have no response to low LET rays, but after the 12 specific gene combinations are irradiated by high LET rays, the gene combinations have obvious high regulation of transcription level, and based on the characteristic biological transcription level change, the gene combinations can be used for identifying different linear energy transfer ray types. The 12 gene markers can also be used for detecting or assisting in detecting lung radiation injury caused by high linear energy transmission rays.
Description
Technical Field
The invention belongs to a radiation biological ray type detection method, and relates to a biological detection marker of heavy ion rays or other high-linear energy transfer (high-LET) rays and application thereof.
Background
Inflammatory responses are a common stressful pathological change of acute, subacute radiation injury. The inflammatory response is mainly secondary to the ionizing radiation induced tissue cell damage and death, resulting in the massive production and release of pro-inflammatory factors (pro-inflammatory factors). Acute inflammation is usually manifested as red, swollen, hot, painful, functional occult, accompanied by fever and leukocytosis. Unlike skin or superficial tissues, radiation-induced lung injury is usually free of significant clinical symptoms during the early stages of inflammation; auxiliary tests including chest radiographs, CT, hematology, pulmonary function tests, etc. also often appear negative. Only in the middle and late stages of the inflammatory response gradually appear signs of irritation, dry cough, associated with shortness of breath, palpitations and chest pain, low fever or high fever. This stage is usually most pronounced starting at 12 weeks (around 3 months) after radiation exposure and is clinically often associated with infectious pneumonia from bacterial infections. When the radioactive pulmonary inflammation after high ionization dose exposure is not effectively controlled, interstitial pulmonary fibrosis and diffuse multiple organ inflammation of the lung can be caused, and the serious pulmonary inflammation can cause respiratory failure and even death.
The explosion time of the atomic bomb lasts only ten seconds, and the bomb is characterized by strong instant lethality. The destruction factors of the nuclear weapons include 5 types, such as thermal radiation (light radiation), shock waves, early nuclear radiation, nuclear electromagnetic pulses, and nuclear radioactive contamination. The first four are transient killer destructive factors generated during the first ten seconds of a nuclear explosion. Radioactive contamination can last for months, years, or longer. The first 4 direct hazards end with the completion of the explosion on the bomb in the island. Thus, radioactive contamination is the only factor that determines whether humans can safely live after a nuclear explosion. After nuclear explosion, mushroom-shaped smoke cloud contains a large amount of radioactive dust, and when the smoke cloud diffuses with wind, the radioactive dust gradually falls to the ground or other objects under the action of gravity or with 'black rain', so that a large radioactive contamination area is formed.
The "radioactive dust" produced by nuclear explosion mainly contains alpha rays, beta rays and gamma rays. That is, a mixed ray of a high linear-energy ray and a low linear-energy ray. This radioactive smoke carrying the mixed radiation can enter lung tissue through the respiratory tract, causing radiation-induced lung injury. Because the body has different stress responses to different types of linear energy transfer rays, namely, the high LET rays with the same dose have larger damage or variation of unrepairable cells than the low LET rays, people can suffer various diseases related to respiratory system, immunity, tumor and hematopoietic tissues after being exposed to the chronic high LET rays. After nuclear radiation accidents and various types of radiation accidents occur, the method for rapidly confirming the type of the exposed rays and detecting the radiation dose is one of the key measures for guiding subsequent prevention and treatment.
Disclosure of Invention
The present invention provides a method for detecting the type of radiation after high LET radiation exposure in the early stages of pulmonary radiation injury inflammation. The method can accurately identify high LET rays according to the mRNA level of the gene marker about 3 weeks after exposure.
The invention aims to provide a high-performance energy-transfer ray radiation marker for detection or auxiliary detection and application thereof.
It is another object of the present invention to provide a product for detecting or aiding in the detection of high linear performance transmitted radiation.
It is a further object of the present invention to provide a product for detecting or aiding in the detection of high linear performance radiation-induced lung injury.
The technical scheme adopted by the invention is as follows:
use of at least one of 12 genes, Retnlb, S100a8, Csf3r, Slc4a1, Tubb1, Clec4d, Il1b, Apol11b, Alas2, Coro1a, Ahsp and Rsad2, as a marker for detecting or aiding in the detection of high linear performance energy transfer radiation.
Application of a reagent for quantitatively detecting at least one gene in 12 genes in preparation of a preparation for detecting or assisting in detecting high linear energy transmission ray radiation, wherein the 12 genes are Retnlb, S100a8, Csf3r, Slc4a1, Tubb1, Clec4d, Il1b, Apol11b, Alas2, Coro1a, Ahsp and Rsad 2.
Furthermore, the reagent for quantitatively detecting at least one gene in 12 genes is a primer or/and a probe for quantitatively detecting at least one gene in 12 genes.
Further, the primer for quantitatively detecting at least one gene in the 12 genes is as follows:
Retnlb：
Retnlb-F：ATGAAGCCTACACTGTGTTTCC
Retnlb-R：CTGCCAGAAGACGTGACACT
S100a8:
S100a8-F：AAATCACCATGCCCTCTACAAG
S100a8-R：CCCACTTTTATCACCATCGCAA
Csf3r:
Csf3r-F：TGCACCCTGACTGGAGTTAC
Csf3r-R：TGAAATCTCGATGTGTCCACAG
Slc4a1:
Slc4a1-F：AGATCCCAGATCGAGACAGC
Slc4a1-R：GCTCCACATAGACCTGACCG
Tubb1:
Tubb1-F：CTGGGAGGTGATCGGGGAA
Tubb1-R：GCACATACTTCTTACCGTAGGCT
Clec4d:
Clec4d-F：ACCCGACATCCCCAACTGAT
Clec4d-R：CTCTCGTCCAGCGTAAAAAGT
Il1b:
Il1b-F：GAAATGCCACCTTTTGACAGTG
Il1b-R：TGGATGCTCTCATCAGGACAG
Apol11b:
Apol11b-F：AAAAGTCATTGATAACGCCACGG
Apol11b-R：CCTCGCTTGACAACTCTACTG
Alas2:
Alas2-F：GCAGCTATGTTGCTACGGTC
Alas2-R：GATGGGGCAGCGTCCAATAC
Coro1a:
Coro1a-F：TGGCTCTGATCTGTGAGGC
Coro1a-R：TCTTGTCTACTCGTCCAGTCTTG
Ahsp:
Ahsp-F：GGATCTCATTTCCGCAGGATTG
Ahsp-R：CTGCTGCCTGTAATAGTTGATGT
Rsad2:
Rsad2-F：AGCATTAGGGTGGCTAGATCC
Rsad2-R：CTGAGTGCTGTTCCCATCTTC。
application of a reagent for quantitatively detecting at least one gene in 12 genes in preparation of a medicament for detecting or assisting in detecting lung injury caused by high linear energy transmission ray radiation, wherein the 12 genes are Retnlb, S100a8, Csf3r, Slc4a1, Tubb1, Clec4d, Il1b, Apol11b, Alas2, Coro1a, Ahsp and Rsad 2.
Furthermore, the reagent for quantitatively detecting at least one gene in 12 genes is a primer or/and a probe for quantitatively detecting at least one gene in 12 genes.
Further, the lung injury is an inflammatory injury.
Further, the high linear energy transfer rays include heavy ion rays, alpha particle rays, fast neutrons, negative pi mesons, helium, carbon, nitrogen, oxygen, neon.
A product for detecting or assisting in detecting high linear energy transmission ray radiation contains a reagent for quantitatively detecting at least one gene in 12 genes, which is a primer or/and a probe for quantitatively detecting at least one gene in 12 genes; the 12 genes are Retnlb, S100a8, Csf3r, Slc4a1, Tubb1, Clec4d, Il1b, Apol11b, Alas2, Coro1a, Ahsp and Rsad 2.
A product for detecting or assisting in detecting lung injury caused by high linear energy transmission ray radiation comprises a reagent for quantitatively detecting at least one gene in 12 genes, wherein the reagent is a primer or/and a probe for quantitatively detecting at least one gene in 12 genes; the 12 genes are Retnlb, S100a8, Csf3r, Slc4a1, Tubb1, Clec4d, Il1b, Apol11b, Alas2, Coro1a, Ahsp and Rsad 2.
Further, the product comprises a kit or a chip.
The invention has the beneficial effects that:
(1) the invention utilizes the whole genome transcriptomics analysis of 3 weeks after mice receive different LET rays and are irradiated on the whole lung to screen out 12 high LET specificity sensitive biological detection markers: retnlb (resistance-Like Beta), S100a8(S100Calcium Binding Protein A8), Csf3r (colloid Stimulating Factor 3Receptor), Slc4a1 (solvent Carrier Family 4Member 1), Tubb1 (tubular Beta 1Class VI), Clec4D (C-Type Lectin Family 4Member D), Il1b (Interleukin 1Beta), Apol11b (Apolipoprotein L1), Alas2(5' -amino Synthnase 2), Coro1A (Coronin 1A), Ahsp Binding Protein, Radisasa 2 (carbon S-Ading 2). The 12 specific gene combinations have no response to low LET rays, but after the 12 specific gene combinations are irradiated by high LET rays, the gene combinations have obvious high regulation of transcription level, and based on the characteristic biological transcription level change, the gene combinations can be used for identifying different linear energy transfer ray types.
(2) The 12 gene markers can be used for detecting or assisting in detecting high-linear energy transfer (high-LET) ray-induced lung radiation injury (early inflammation stage).
(3) The 12 radiation-sensitive gene combinations disclosed by the invention have no response to low LET (such as X-ray) radiation, but after receiving high LET radiation, the sensitive gene combinations have obviously high-regulated transcription levels. Whether the LET ray exposure is high can be determined according to the average value of the transcription level difference expression fold of the 12 specific X ray sensitive genes (Retnlb, S100a8, Csf3r, Slc4a1, Tubb1, Clec4d, Il1b, Apol11b, Alas2, Coro1a, Ahsp and Rsad 2).
Drawings
FIG. 1: compared with a blank control group (0Gy) at 3 weeks after the C57BL/6 mouse is irradiated with heavy ion rays of 12.5Gy, dose-dependent up-regulated gene subsets are respectively obtained after the whole genome transcription level of lung tissues is analyzed, and volcano map differential gene analysis is carried out.
FIG. 2: the volcano images of the heavy ion radiation group are subjected to KEGG signal channel enrichment analysis to obtain differential genes, which indicates that the differential genes have high correlation with biological processes such as cytokine-cytokine receptor interaction, chemokine signal channel, influenza A, systemic lupus erythematosus, hematopoietic cell lineage, leishmaniasis, rheumatoid arthritis, p53 signal channel, staphylococcus aureus infection, prion and the like.
FIG. 3: 3 weeks after the C57BL/6 mouse is irradiated by heavy ion rays of 12.5Gy (namely, the biological equivalent dose of 20GyE) or X rays of 20Gy, the lung tissue whole genome transcription level is analyzed, and then dose-dependent up-regulated gene subsets are respectively obtained for volcano-map differential gene analysis.
FIG. 4: the heavy ion beam relative blank contrast high-level differential gene subset (figure 1) and the heavy ion beam relative X-ray high-level differential gene subset (figure 3) were analyzed by a Venn diagram to obtain 67 heat maps of the differentially expressed genes. The gene set was up-regulated 3 weeks after irradiation with heavy ion beam, but slightly up-regulated or without significant change after X-ray irradiation.
FIG. 5: screening to obtain heatmaps of 12 specific sensitive genes. The gene combination can effectively prompt that no obvious expression difference exists when normal lung tissues are irradiated by X rays or low LET rays, but the expression is obviously up-regulated when heavy ion rays are irradiated.
FIG. 6: qRT-PCR detection shows that 12 specific sensitive genes of the present invention have gene expression difference multiple (log2fold change) after being irradiated by X ray or heavy ion ray for 3 weeks. The specific gene combination can be obviously up-regulated and expressed after heavy ion radiation, and has no obvious change after X-ray radiation.
FIG. 7: the average value (log2 relative expression value) of 12 specific sensitive genes in a blank control group, an X-ray irradiation group and a heavy ion ray irradiation group is obtained. The mean value of the gene expression after heavy ion ray irradiation is obviously higher than that of a blank control (P <0.01) and an X-ray irradiation group (P < 0.05).
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
The method comprises the following steps:
1. animal experiment irradiation and grouping: c57BL/6 female mice 8-10 weeks old are bred in SPF grade under the conditions of temperature (23 +/-1) DEG C, relative humidity 55 +/-5%, pressure difference less than or equal to 10Pa, light irradiation for 12h every day and free drinking water.
2. After isoflurane induced anesthesia before irradiation, the mice are transferred to a special mouse chest irradiation device and fixed properly. The whole lung field irradiation is carried out by adopting X rays and heavy ion (carbon ion) rays respectively, and lung injury inflammation (early inflammation stage) is caused.
3. The irradiation and grouping include a blank control group (0Gy), a heavy ion ray single 12.5Gy irradiation group, and an X ray single 20Gy irradiation group.
4. Fresh lung tissue was extracted 3 weeks after irradiation, treated with total RNA extraction reagent (Trizol), and total RNA was isolated and extracted using RNeasy Mini kit. To avoid DNA contamination, RNA was treated with DNase I. Purified RNA was eluted in 45.0. mu.l nuclease-free water and stored at-20.0 ℃. RNA concentration and purity were assessed using a NanoDrop ND-1000 spectrophotometer. The integrity and purity of the RNA samples were determined using a 2100Bioanalyzer and the corresponding RNA Nano Chip. Samples were analyzed based on whole genome gene expression arrays. And generating an expression matrix by using a self-contained software package, and performing data annotation and summarization. In subsequent statistical operations, the data were log2 transformed to account for the increase or decrease in gene expression. In order to analyze the relation between radiation and gene expression trend at different time points, R language software package is adopted for carrying out cluster analysis, including principal component analysis, hierarchical cluster, K mean cluster and self-organizing mapping to identify the gene expression related to the dosage.
As a result:
FIG. 1 is a volcano diagram of dose-dependent up-regulated expression gene subsets obtained after the lung tissue whole genome transcription level analysis and differential gene analysis of C57BL/6 mice subjected to 12.5Gy heavy ion radiation in 3 weeks compared with a blank control group (0 Gy). After being irradiated by heavy ion rays of 12.5Gy, 131 genes with the up-regulation expression multiple larger than 1.5 times are present, and 11 genes with the down-regulation expression multiple larger than 1.5 times are present.
FIG. 2 is a KEGG signal pathway enrichment analysis of genes differentially expressed from heavy ion-irradiated volcano maps, suggesting a high correlation with cytokine-cytokine receptor interactions, chemokine signal pathways, influenza A, systemic lupus erythematosus, hematopoietic lineage, leishmaniasis, rheumatoid arthritis, p53 signal pathways, Staphylococcus aureus infection, prions, and other biological processes.
FIG. 3 shows that 3 weeks after C57BL/6 mice are irradiated with heavy ion dose of 12.5Gy (i.e. biological equivalent dose of 20GyE) and X-ray dose of 20Gy, the lung tissue whole genome transcript level analysis is carried out to obtain dose-dependent up-regulated gene subsets, and volcano-map differential expression gene analysis is carried out. As can be seen from the figure, there were 84 genes whose expression factor was up-regulated by more than 1.2 in the heavy ionizing radiation group and 82 genes whose expression factor was down-regulated by more than 1.2 in the X-ray radiation group.
FIG. 4 is a heat map of 67 up-regulated expression genes obtained by Venn map analysis of the subset of differential genes up-regulated in the heavy ion beam set of FIG. 1 relative to the blank control set and the subset of differential genes up-regulated in the heavy ion beam radiation set of FIG. 3 relative to the X-ray radiation set. The 67 gene sets were up-regulated in expression 3 weeks after heavy ion beam irradiation, but no up-regulated expression or insignificant up-regulated expression was observed after X-ray irradiation, i.e., the 67 genes were heavy ion beam-specific sensitive genes.
The invention further analyzes, studies and screens the 67 genes, and finally determines 12 target genes, namely 12 genes of Retnlb, S100a8, Csf3r, Slc4a1, Tubb1, Clec4d, Il1b, Apol11b, Alas2, Coro1a, Ahsp and Rsad 2. FIG. 5 is a heat map analysis of the gene expression levels of lung tissues of mice after irradiation with different rays, respectively, showing that the 12 gene expression levels are significantly up-regulated at a time point of 3 weeks after irradiation with heavy ion rays; without significant up-regulation of expression by X-ray radiation. The 12 gene combinations can effectively indicate that normal lung tissues have no obvious expression difference when being irradiated by X rays or low LET rays, but have obvious up-regulation expression when being irradiated by heavy ion rays.
The 12 genes thus identified were further subjected to qRT-PCR, and the expression levels of the 12 genes in lung tissues of each group (blank, 20GyX radiation, 12.5Gy heavy ion radiation) were determined. The detection results are shown in fig. 6, and it can be seen from the results that the differences in gene expression values of 12 genes of the present invention after X-ray and heavy ion ray irradiation significantly up-regulated in the heavy ion ray irradiation group, whereas in X-ray, the 12 genes were not significantly up-regulated. FIG. 7 is a further statistical analysis of the mean value of fold difference in expression of 12 genes from each group in FIG. 6, which shows that the mean value of fold difference in gene expression after irradiation with X-ray and heavy ion beam is statistically significant (P <0.01, P <0.05) for both the heavy ion beam group and the blank group and the heavy ion beam group and the X-ray group.
The primers for detecting the 12 genes of the invention by the line qRT-PCR are respectively as follows:
Retnlb：
Retnlb-F：ATGAAGCCTACACTGTGTTTCC(SEQ ID NO:1)
Retnlb-R：CTGCCAGAAGACGTGACACT(SEQ ID NO:2)
S100a8:
S100a8-F：AAATCACCATGCCCTCTACAAG(SEQ ID NO:3)
S100a8-R：CCCACTTTTATCACCATCGCAA(SEQ ID NO:4)
Csf3r:
Csf3r-F：TGCACCCTGACTGGAGTTAC(SEQ ID NO:5)
Csf3r-R：TGAAATCTCGATGTGTCCACAG(SEQ ID NO:6)
Slc4a1:
Slc4a1-F：AGATCCCAGATCGAGACAGC(SEQ ID NO:7)
Slc4a1-R：GCTCCACATAGACCTGACCG(SEQ ID NO:8)
Tubb1:
Tubb1-F：CTGGGAGGTGATCGGGGAA(SEQ ID NO:9)
Tubb1-R：GCACATACTTCTTACCGTAGGCT(SEQ ID NO:10)
Clec4d:
Clec4d-F：ACCCGACATCCCCAACTGAT(SEQ ID NO:11)
Clec4d-R：CTCTCGTCCAGCGTAAAAAGT(SEQ ID NO:12)
Il1b:
Il1b-F：GAAATGCCACCTTTTGACAGTG(SEQ ID NO:13)
Il1b-R：TGGATGCTCTCATCAGGACAG(SEQ ID NO:14)
Apol11b:
Apol11b-F：AAAAGTCATTGATAACGCCACGG(SEQ ID NO:15)
Apol11b-R：CCTCGCTTGACAACTCTACTG(SEQ ID NO:16)
Alas2:
Alas2-F：GCAGCTATGTTGCTACGGTC(SEQ ID NO:17)
Alas2-R：GATGGGGCAGCGTCCAATAC(SEQ ID NO:18)
Coro1a:
Coro1a-F：TGGCTCTGATCTGTGAGGC(SEQ ID NO:19)
Coro1a-R：TCTTGTCTACTCGTCCAGTCTTG(SEQ ID NO:20)
Ahsp:
Ahsp-F：GGATCTCATTTCCGCAGGATTG(SEQ ID NO:21)
Ahsp-R：CTGCTGCCTGTAATAGTTGATGT(SEQ ID NO:22)
Rsad2:
Rsad2-F：AGCATTAGGGTGGCTAGATCC(SEQ ID NO:23)
Rsad2-R：CTGAGTGCTGTTCCCATCTTC(SEQ ID NO:24)。
in summary, the present invention provides a target for detecting early stage inflammation of lung radiation injury, a detection method based on the above-mentioned 12 specific gene marker expression level changes, and identifies whether the object is damaged by high-linear energy transfer (high-LET) rays. The invention screens out the high LET specific sensitive gene combination by utilizing the whole genome transcriptomics analysis of 3 weeks after the mice receive the low and high LET rays and are irradiated on the whole lungs. The specific gene combination has no obvious response to low LET rays (such as X rays), but after the specific gene combination is irradiated by high LET rays, the gene combination has obvious up-regulated expression of transcription level, and based on the change of the specific biological transcription level, the gene combination can also be used for identifying different linear energy transfer ray types.
The invention discovers that a rapid biological dose evaluation means can be established according to the stress change of the transcription level of the relevant radiation sensitive genes of the lung tissues of mice after high-linear energy transfer (high-LET) rays are exposed acutely. The gene marker contained in the detection method has high specific radiation sensitivity to high LET rays (such as heavy ion rays and alpha particles) and no radiation sensitivity to low LET rays (such as X rays), so that the gene marker can be used for identifying acute high LET ray radiation exposure of normal tissues and providing a radiation reference dose.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> southern medical university
<120> high-linear-performance biological detection marker for transmitting ray radiation and application thereof
<130>
<160> 24
<170> PatentIn version 3.5
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Claims (3)
1. Application of a reagent for quantitatively detecting expression levels of 11 genes in preparation of a preparation for detecting lung injury inflammation caused by heavy ion ray radiation, wherein the 11 genes are Retnlb, S100a8, Csf3r, Slc4a1, Tubb1, Clec4d, Il1b, Apol11b, Alas2, Coro1a and Rsad2, and the lung injury inflammation is in an early stage of inflammation.
2. The use of claim 1, wherein the primers for quantitatively detecting the expression levels of 11 genes are:
Retnlb：
Retnlb-F：ATGAAGCCTACACTGTGTTTCC
Retnlb-R：CTGCCAGAAGACGTGACACT
S100a8:
S100a8-F：AAATCACCATGCCCTCTACAAG
S100a8-R：CCCACTTTTATCACCATCGCAA
Csf3r:
Csf3r-F：TGCACCCTGACTGGAGTTAC
Csf3r-R：TGAAATCTCGATGTGTCCACAG
Slc4a1:
Slc4a1-F：AGATCCCAGATCGAGACAGC
Slc4a1-R：GCTCCACATAGACCTGACCG
Tubb1:
Tubb1-F：CTGGGAGGTGATCGGGGAA
Tubb1-R：GCACATACTTCTTACCGTAGGCT
Clec4d:
Clec4d-F：ACCCGACATCCCCAACTGAT
Clec4d-R：CTCTCGTCCAGCGTAAAAAGT
Il1b:
Il1b-F：GAAATGCCACCTTTTGACAGTG
Il1b-R：TGGATGCTCTCATCAGGACAG
Apol11b:
Apol11b-F：AAAAGTCATTGATAACGCCACGG
Apol11b-R：CCTCGCTTGACAACTCTACTG
Alas2:
Alas2-F：GCAGCTATGTTGCTACGGTC
Alas2-R：GATGGGGCAGCGTCCAATAC
Coro1a:
Coro1a-F：TGGCTCTGATCTGTGAGGC
Coro1a-R：TCTTGTCTACTCGTCCAGTCTTG
Rsad2:
Rsad2-F：AGCATTAGGGTGGCTAGATCC
Rsad2-R：CTGAGTGCTGTTCCCATCTTC。
3. a product for detecting lung injury inflammation caused by heavy ion ray radiation is characterized in that the product contains primers for quantitatively detecting the expression levels of 11 genes; the 11 genes are Retnlb, S100a8, Csf3r, Slc4a1, Tubb1, Clec4d, Il1b, Apol11b, Alas2, Coro1a, Ahsp and Rsad 2; the lung injury inflammation is an early inflammatory stage; the primers for quantitatively detecting the expression levels of 11 genes are as follows:
Retnlb：
Retnlb-F：ATGAAGCCTACACTGTGTTTCC
Retnlb-R：CTGCCAGAAGACGTGACACT
S100a8:
S100a8-F：AAATCACCATGCCCTCTACAAG
S100a8-R：CCCACTTTTATCACCATCGCAA
Csf3r:
Csf3r-F：TGCACCCTGACTGGAGTTAC
Csf3r-R：TGAAATCTCGATGTGTCCACAG
Slc4a1:
Slc4a1-F：AGATCCCAGATCGAGACAGC
Slc4a1-R：GCTCCACATAGACCTGACCG
Tubb1:
Tubb1-F：CTGGGAGGTGATCGGGGAA
Tubb1-R：GCACATACTTCTTACCGTAGGCT
Clec4d:
Clec4d-F：ACCCGACATCCCCAACTGAT
Clec4d-R：CTCTCGTCCAGCGTAAAAAGT
Il1b:
Il1b-F：GAAATGCCACCTTTTGACAGTG
Il1b-R：TGGATGCTCTCATCAGGACAG
Apol11b:
Apol11b-F：AAAAGTCATTGATAACGCCACGG
Apol11b-R：CCTCGCTTGACAACTCTACTG
Alas2:
Alas2-F：GCAGCTATGTTGCTACGGTC
Alas2-R：GATGGGGCAGCGTCCAATAC
Coro1a:
Coro1a-F：TGGCTCTGATCTGTGAGGC
Coro1a-R：TCTTGTCTACTCGTCCAGTCTTG
Rsad2:
Rsad2-F：AGCATTAGGGTGGCTAGATCC
Rsad2-R：CTGAGTGCTGTTCCCATCTTC。
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