CN110890133A

CN110890133A - Application of acute response phase protein SAA1 in construction of post-ionizing radiation lethality prediction model or preparation of kit and reagent

Info

Publication number: CN110890133A
Application number: CN201911141481.7A
Authority: CN
Inventors: 王治东; 林仲武; 王琪; 黄金凤; 戚振华
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2019-11-20
Filing date: 2019-11-20
Publication date: 2020-03-17
Anticipated expiration: 2039-11-20
Also published as: CN110890133B

Abstract

Application of acute reaction phase protein SAA1 in constructing a post-ionizing radiation lethality prediction model or preparing a kit and a reagent, and belongs to the technical field of nuclear radiation damage detection. In order to solve the problem of how to predict or evaluate the radiation lethality of an individual to be tested, the invention provides an application of secondary increase of the content of protein SAA1 in an in-vivo acute reaction period after the individual to be tested receives ionizing radiation as a lethality index in evaluating the lethality of the individual to be tested after receiving the ionizing radiation, such as building an evaluation prediction model, a related product kit or a reagent. In addition, the invention also proves that the SAA1 or SAA1 can detect the irradiated dose of the irradiated individual or distinguish the irradiated dose range by combining the peripheral blood lymphocyte number, and can be used for preparing related detection kits or reagents. The invention is beneficial to the evaluation and detection of radiation damage.

Description

Application of acute response phase protein SAA1 in construction of post-ionizing radiation lethality prediction model or preparation of kit and reagent

Technical Field

The invention belongs to the technical field of nuclear radiation damage detection, and particularly relates to an application of an acute reaction phase protein SAA1 in construction of a post-ionizing radiation lethality prediction model or preparation of a kit and a reagent.

Background

Conditions such as nuclear terrorist attacks, nuclear power station accidents, radioactive source accidents, etc. can cause acute radiation damage to the human body in such environments. Different degrees of radiation injury adopt different treatment schemes, for example, mild radiation injury is gradually recovered through supporting treatment modes such as nutrition and rest, and lethal radiation needs to be treated through bone marrow transplantation and the like. How to judge whether a person is irradiated and whether radiation damage to the irradiated person is fatal!

SAA1 is an acute response phase protein that can be elevated about ten-fold in local or mild inflammation and hundreds of-fold in the presence of severe infections in the body, SAA1 is synthesized primarily by the liver, and is induced by exogenous Toll-like receptor ligands such as LPS, and endogenous cytokines such as interleukin 1 β, interleukin 6, and tumor necrosis factor, in addition, SAA1 can be detected in body tissues other than the liver, such as breast tissue, colon, esophagus, kidney, large intestine, pituitary, and spleen, SAA1 encoded by the SAA1 gene is highly similar in humans and mice and plays a role in high density lipoprotein remodeling, lipid metabolism, antibacterial infection, immune regulation, and tumor pathology.

In the event of a radiological event, extensive population screening is required to classify the exposed individuals, and subsequent exposure dose assessment is an important preliminary screen for emergency victims of massive casualties. Depending on the exposure dose, some require only supportive care, while for high dose exposures medical intervention such as stem cell transplantation is required. Thus, for the evaluation of the exposure dose, it can be determined whether the exposure dose of the patient requires carefully deployed medical resources and care.

The identification of severely damaged individuals exposed to the same dose of radiation remains a challenge in estimating the radiation dose, patent application CN 110308285a discloses that the acute response phase protein SAA in combination with IL-1 α as a combined marker can be used to estimate the exposure dose of the individual to be tested, but the combined marker is difficult to be used to predict the mortality rate because the combined marker has no difference between the death group and the survival group.

Disclosure of Invention

Aiming at the problem of how to predict or evaluate the radiation lethality of an individual to be tested, the invention provides application of an acute response period protein SAA1 in constructing a post-ionizing radiation lethality prediction model.

Further limiting, the dependent variable of the prediction model is a lethal index, and the lethal index is a secondary increase of the content of the protein SAA1 in the acute reaction phase in the body after the individual to be tested receives ionizing radiation.

Further defined, the secondary increase refers to a decrease after the content of the protein SAA1 in the acute response phase in vivo is increased for the first time and then is increased rapidly again after the subject to be tested receives ionizing radiation.

Further, it is defined that the intensity of the ionizing radiation is 8Gy or more.

The invention also provides application of the acute response phase protein SAA1 in preparing a kit or reagent for evaluating or predicting lethality after ionizing radiation.

Further limited, the content of the acute response phase protein SAA1 in the test subject is increased twice after being irradiated, and the increase is used as the target detection index of the kit.

Further defined, the secondary increase is a decrease after the content of the protein SAA1 in the acute response phase in vivo is increased after the subject to be tested receives ionizing radiation, and then the protein SAA1 is rapidly increased again.

Further defined, the SAA1 alone can be used as a detection target to obtain an assessment or prediction result.

The invention also provides application of the acute response phase protein SAA1 in preparation of a kit or a reagent for evaluating the efficacy and/or prognosis of an ionizing radiation medicament.

Further, the intensity of the ionizing radiation is 8Gy or more.

In a further definition, the medicament is for the prevention or treatment of ionizing radiation.

In a further definition, the drug is selected from small molecule polypeptides.

Further defined, the acute response phase protein SAA1 alone was used as the target protein for evaluation.

In order to solve the problem of how to detect the irradiated dose of the irradiated individual, the invention also provides application of the acute response phase protein SAA1 in preparing a kit or a reagent for detecting the irradiated dose after ionizing radiation or distinguishing the irradiated dose range.

The invention also provides application of the combination of the acute response phase protein SAA1 and the number of peripheral blood lymphocytes in the preparation of a kit or a reagent for detecting the irradiated dose or distinguishing the irradiated dose range after ionizing radiation.

Advantageous effects

The invention establishes a mouse radiation model, evaluates the change of SAA1 expression in response to whole body and part body radiation at early stage, performs whole body irradiation with different doses by taking a C57BL/6J mouse as an animal model, and adds amifostine for protection; nasopharyngeal carcinoma patients receiving radiation therapy were used as a human irradiated model. The results show that the serum of the mice of each dose irradiation group shows different increases of the SAA1 in 6 hours, reaches a peak value in 12 hours, is reduced in 3 days, the serum SAA1 level is observed to be increased for the second time from 5 days to 7 days, the second increase is related to the subsequent death condition of the mice, the increase of the SAA1 is also detected in the human serum of the nasopharyngeal carcinoma patients after the radiation treatment, the experiment proves the feasibility of the SAA1 alone as a biomarker in the radiation exposure of the human body, and the second increase of the SAA1 after the individuals to be detected receive the radiation can be used as an index for predicting the radiation lethality, and the experiment can be used for constructing a model for predicting the lethality after the ionizing radiation and predicting the radiation lethality. The invention also demonstrates that the combination of SAA1 or SAA1 with peripheral blood lymphocyte number can estimate the radiation dose of the irradiated individual, classify the dose range and help to identify the damaged individual.

Drawings

FIG. 1. 30-day survival assay, body weight assay and peripheral blood cell assay for different dose systemically irradiated mice, where a is the 30-day survival of 0,1, 2,4, 8 and 12Gy irradiated female C57BL/6J mice; b is the body weight of female C57BL/6J mice irradiated at different times with 0,1, 2,4, 8 and 12 Gy; c-f are the number of lymphocytes, erythrocytes, platelets and leukocytes varied at different times of 0,1, 2,4, 8 and 12Gy irradiation of female C57BL/6J mice, respectively, with n-8 per group;

FIG. 2 time and dose response of SAA1 in mouse serum after different doses of whole body irradiation, where a is the SAA1 concentration at 0.125, 0.5,1,2,3,5, and 7 days after irradiation in 0,1, 2,4, 8, and 12Gy irradiated female C57BL/6J mice; (b)0.25, (c)0.5, (d)1, (e)2, (f)3, (g)5 and (h)7 days post exposure to radiation, respectively, as a function of dose, SAA1 concentration, each black dot representing one animal, each group n-8;

FIG. 3 analysis of SAA1 concentration in mouse serum after irradiation and systemic infection parameters, wherein a is

day

0,1,5 and 7 after 8Gy systemic irradiation, SAA1 and LPS in serum and 16S rRNA expression in liver of 31 mice (7 mice on

day

7, 8 mice in each group on

days

0,1 and 5, No.1-No.8 in the figure); b is the SAA1 and PCT concentrations in the serum of 23 mice (2 mice on

day

7,3 mice in each group on days 0, 0.125, 0.5,1,2,3,5, day 7, No.1-No.3 in the figure) on days 0, 0.125, 0.5,1,2,3,5 and 7 after 12Gy total body irradiation;

FIG. 4 serum SAA1 changes in partially irradiated miceIn case a is liver screening in PBI-1 and PBI-3 of C57BL/6J mice, exposing liver in PBI-2 and PBI-4 groups; b is the concentration of SAA1 in serum and (c) mRNA expression in liver at 12 hours after 8Gy irradiation in control, whole body irradiation and PBI groups, each group n-6, compared to control,^*P＜ 0.05，^**P＜0.01，^***p < 0.001 and^****P＜0.0001；

FIG. 5. Effect of radiation treatment on serum SAA1 from patients with nasopharyngeal carcinoma, (a) scattergrams showing SAA1 concentrations (P < 0.0001) before and after radiation treatment for 17 cases of nasopharyngeal carcinoma; (b) SAA1 was used as a ROC curve for predicting radiation exposure biomarkers for nasopharyngeal carcinoma patients.

Detailed Description

The medicines, reagents, instruments, equipment and the like used in the invention can be purchased through commercial approaches if no special instructions exist, and the experimental methods are all conventional methods in the field if no special instructions exist, or the experimental methods are performed by referring to corresponding instructions.

The experimental animals used in the following examples were: female C57BL/6J mice (6-8 weeks old) were purchased from Experimental animals technology, Inc., Vitonuli, Beijing, and bred at the military medical research institute (Beijing), military academy of sciences. Mice were housed in uniform cages at a controlled temperature, humidity, and light/dark cycle of 12/12 hours in the presence of a particular pathogen-free medium, and were raised for one week prior to the experiment and weighed 19-21 g. Animal care and handling was performed according to the "chinese AMMS laboratory animal care and use guidelines", all animal experiments approved by the animal care and use committee of the beijing radiology institute (beijing).

The nucleotide sequence of the acute response phase protein SAA1 and the murine SAA1 is shown as SEQ ID No. 1; the amino acid sequence is shown as SEQ ID No. 2; the nucleotide sequence of the human SAA1 is shown as SEQ ID No. 3; the amino acid sequence is shown as SEQ ID No. 4.

Enzyme-linked immunosorbent assay (ELISA) kit is used for detecting SAA1 and Procalcitonin (PCT), wherein (product information of mouse SAA1 ELISA kit: EK1190, BOSTER, Wuhan, China; product information of human SAA1 ELISA kit: EK1544, BOSTER, Wuhan, China; product information of PCT ELISA kit: E1071M, Cuxabo, Wuhan, China, and operation method is as per the instruction.

Limulus reagent product information for lipopolysaccharide assay EC80545, biondo, Xiamen, China.

TRIzol product information Invitrogen, Carlsbad, CA, USA.

PrimeScript RT kit product information Takara, Shiga, Japan.

iTaq Universal SYBR Green Supermix product information BioRad, Rachmond, Calif., USA.

The information of the Tiangen genome DNA kit product includes Tiangen, Beijing and China.

Example 1 animal model irradiation experiments.

1. Whole body irradiation (TBI), local irradiation and sample collection

In the time and dose response study experiments, mice were placed in plexiglas transparent boxes to prevent movement and irradiated with a cobalt-60 radiation source at a dose rate of 85.08cGy/min at single irradiation doses of 0,1, 2,4, 8 and 12 Gy.

At various time points (0.25, 0.5,1,2,3,5 and 7 days) after irradiation, whole blood was removed from the eye and the mice were sacrificed by decapitation. Under the same irradiation conditions as described above, another group of mice collected 20. mu.L of blood from the tail vein, analyzed for blood cell changes, weighed 1,3, 7, 11, 15, 20, 25 and 30 days after irradiation, and observed for survival.

In the study of the experiment for dynamic monitoring of the concentration of SAA1, the mouse is randomly bound with the ear ring marked with random numbers, the mouse is raised for 5 days after the ear ring is bound until the normal state is recovered, the mouse in 10Gy irradiation group is placed in an organic glass transparent box and receives 73.02cGy/min of dosage rate⁶⁰The gamma-ray irradiation of Co source is carried out, the total dose is 10Gy, each group of mice collects venous blood with the dose of about 40 mu L from tail vein respectively 4 days before irradiation and 1,3,5 and 7 days after irradiation, and the survival condition of each group of mice within 30 days after irradiation is observed.

Local irradiation (PBI) mice were anesthetized by intraperitoneal injection of 10mg/kg sodium pentobarbital, and then placed in an organic glass box at a uniform position, and the non-irradiated parts were shielded with lead bricks to expose only the desired body parts for irradiation.

Separating serum, standing whole blood at 4 deg.C for 1 hr, centrifuging at 5000rpm/min at 4 deg.C for 5 min, and storing at-80 deg.C.

2. Amifostine treated mice

Randomly binding earrings marked with random numbers on mice, raising the mice for 5 days after binding the earrings until the mice are recovered to a normal state, injecting 150mg/kg of amifostine into the abdominal cavity 0.5 hour before irradiation, collecting venous blood with the dose of about 40 mu L from the tail vein 4 days before irradiation and 1,3,5 and 7 days after irradiation, and observing the survival condition of each group of mice within 30 days after irradiation.

3.ELISA

SAA1 and Procalcitonin (PCT) were performed using an enzyme-linked immunosorbent assay (ELISA) kit as described in the manual.

LPS detection

The activity of lipopolysaccharide in serum was measured using a limulus reagent for lipopolysaccharide assay. mu.L of serum was diluted in 90. mu.L of the sample treatment solution, and after dilution in a heat-set test tube, the tube was placed in a 70 ℃ water bath for 10 minutes and then in an ice bath for 3 minutes. The concentration of LPS in serum samples was determined using a standard endotoxin standard curve.

Isolation and quantitative PCR analysis of DNA

An appropriate amount of liver was cut into pieces and placed in an EP tube containing 200. mu.L of physiological saline. Total DNA was isolated using the Tiangen genomic DNA kit according to the manufacturer's instructions. Using 16S rRNA gene targeting primers, forward primer: AACGCGAAGAACCTTAC, reverse primer: CGGTGTGTACAAGACCC, quantitative PCR was performed using iTaq Universal SYBR GreenSupermix on a BIORAD CFX96 instrument.

Statistical analysis was performed using IBM SPSS 21. And (4) carrying out t test on the normally distributed data to evaluate the difference between two groups, wherein the non-normally distributed data adopts non-parametric test. Serum SAA1 levels were statistically analyzed by multiple linear regression analysis as a predictor of irradiated dose feasibility following whole body irradiation. A nonparametric Spman rank correlation method is adopted to carry out binary correlation analysis on the SAA1, LPS and 16S rRNA of 8Gy irradiated mice, and carry out correlation analysis on the SAA1 and PCT of 12Gy irradiated mice. Determining a predictive cutoff value from a Receiver Operating Characteristic (ROC) curve; the survival distribution is determined by Log-rank test; mann Whitney u was used to test the concentration of SAA1 in patients before and after radiation therapy.

The results are as follows:

1. mouse survival, body weight and peripheral blood cell number changes after different doses of irradiation

Mice were irradiated at various doses such as 0,1, 2,4, 8 and 12Gy, and observed for changes in survival rate, body weight and peripheral blood cell count of the mice after irradiation. As shown in a in fig. 1, after 0,1, 2, and 4Gy irradiation, the survival rate of the mice was 100% in 30 days, the survival rate of the mice was 0% in 30 days after 12Gy irradiation, 5 mice died and 3 mice survived in 30 days after 8Gy irradiation. Changes in body weight and lymphocyte indices were time and dose dependent (b-f in FIG. 1).

2. Increase in serum SAA1 concentration in C57BL/6J mice by whole body irradiation

To evaluate the time and dose effects of serum SAA1 concentration after total body irradiation of mice, serum SAA1 concentration was measured by ELISA method at 0.25, 0.5,1,2,3,5 and 7 days after total body irradiation of C57BL/6J mice with 1,2,4, 8 and 12 Gy. In fig. 2 a shows that the SAA1 concentration in each dose group began to increase at 6 hours after the irradiation, peaked at 12 hours, and then substantially returned to the initial level on day 3. In FIG. 2 b, c, d, e, f, g and h show the concentration of SAA1 at 0.25, 0.5,1,2,3,5 and 7 days after each dose. On day 5 post-irradiation, 2 of 8Gy groups of 8 mice exhibited a substantial re-increase in SAA1 (60.66. mu.g/ml and 113.26. mu.g/ml, units are the same below), and 6 of 8 mice in 12Gy groups exhibited a re-increase in SAA1 (109.62,301.87,365.43,424.15,332.16,284.66. mu.g/ml). On day 7 post-exposure, 4 of 8 mice in the 8Gy group had a large re-increase in SAA1 (156.79,106.12,170.22,107.31. mu.g/ml), and 2 mice surviving in the 12Gy group had a large increase in SAA1 (234.23, 114.54. mu.g/ml).

3. Elevation of serum SAA1 following irradiation is not caused by infection

Infection is one of the symptoms of acute radiation disease after systemic exposure, and this section of the experiment explored whether elevation of SAA1 was caused by systemic infection. As a direct factor of bacterial infection, LPS is an endotoxin in gram-negative bacteria, and 16S rRNA is a DNA sequence corresponding to rRNA encoded by bacteria, and is present in the genomes of all bacteria. Bacterial translocation was determined as the bacterial load in liver tissue and could be quantified by quantitative PCR using the 16S rRNA gene. In the 8Gy whole body irradiation group we measured serum LPS activity and liver 16S rRNA expression of each of 31 mice on

days

0,1,5 and 7 (7 mice per group on day 7). The results show (a in FIG. 3) that no one-to-one correspondence was observed in serum SAA1 level, LPS activity and liver 16S rRNA expression level in the same mouse. Taking the parameters after 7 days of 8Gy irradiation as examples, 7 mice had SAA1 concentrations of 156.79, 0.12, 18.92, 0.51, 106.12,170.22, and 107.31 μ g/ml, LPS levels of 0.97,0.03,0.18,1.56,0.03,0.06, and 0.98EU/ml, and fold changes in the liver 16S rRNA: 1.14,4.03,1.73,1.28,2.39,3.44 and 3.39 times. In addition to measuring direct indicators of bacterial infection (LPS and 16SrRNA), we selected partial 12Gy whole body irradiated mice (2 mice on

day

7,3 mice each on days 0, 0.25, 0.5,1,2,3, 5), analyzed the levels of serum PCT concentrations (markers of bacterial infection) at day 0, 0.25, 0.5,1,2,3,5, 7 after irradiation, and analyzed their correlation with the level of SAA1 expression. Results as shown in b in fig. 3, PCT levels did not correlate with SAA1 expression levels. For example, mice (3 each) had SAA1 concentrations of 38.26, 28.40, 40.11. mu.g/ml and 109.62,301.87, 0.25. mu.g/ml on day 1 and day 5, respectively, while PCT concentrations on day 1 and day 5 were 0.14, 0.16, 0.12ng/ml and 0.25, 0.13, 0.15ng/ml, respectively.

The late progression and symptoms of acute radiation disease are very similar to classical infections. LPS as an exogenous Toll-like receptor ligand inducing SAA1 and PCT are biomarkers of bacteremia. The 16S rRNA gene is highly conserved in bacteria and is a standard method for identifying and classifying prokaryotes. According to the invention, SAA1, PCT and LPS are measured in the same serum, 16S rRNA expression is evaluated in the liver of the same mouse, and the result of bivariate correlation analysis shows that the concentration of SAA1 in the serum of the 8Gy systemically irradiated mouse has no correlation with LPS activity and liver 16S rRNA expression. There was no correlation between 12Gy whole body irradiated mouse serum SAA1 and PCT concentrations, i.e., increased SAA1 was not associated with infection.

4. SAA1 Change after local irradiation

In radiation accidents, partial exposure to radiation is more common than whole body exposure. As shown above, liver is the major source of SAA1 after exposure, and the variation of SAA1 in mice exposed to shielded liver conditions is unclear. To investigate the varying levels of SAA1 under liver-shielded conditions, animals were irradiated with four localized irradiation patterns, exposing the head and upper thorax (PBI-1), shielding the head and upper thorax (PBI-4), shielding the posterior flank and hind legs (PBI-2), and exposing the posterior flank and hind legs (PBI-3), as shown in fig. 4 a, with the only difference between PBI-1 and PBI-2 being that the liver was shielded in PBI-1 and not in PBI-2; similarly, in PBI-3 the liver was masked, while in PBI-4 it was not. In FIG. 4, b, c show the mRNA level changes of SAA1 in serum and SAA1 in liver at 12h after 8Gy irradiation. Compared with the control group, the fold of the liver exposed group (PBI-2 and PBI-4) is higher than that of the liver unexposed group (PBI-1 and PBI-3). To verify whether the SAA1 concentration prediction global exposure dose threshold at 0.5 days post exposure is equally applicable to localized exposure, a calculation control and PBI group (PBI-1, PBI-2, PBI-3, PBI-4) of 3.26 μ g/ml were selected to obtain a sensitivity of 22/24, specificity of 24/24 and accuracy of 24/24. Thus, it can be demonstrated that the threshold concentration of SAA1 at 0.5 days post total body irradiation is also applicable to local irradiation. Then, to investigate whether SAA1 could be used as a biomarker for mouse PBI exposure area, a subject-operator curve (ROC) was used to distinguish between large area liver local exposure (PBI-2 and PBI-4) and shielded liver small area local exposure (PBI-1 and PBI-3) resulting in a cut-off of 11.46 μ g/ml, sensitivity 12/12, specificity 11/12, and accuracy 12/13.

5, predicting whole body irradiation dose of mice by SAA1 and distinguishing irradiation dose range

To investigate whether serum SAA1 levels could be used as a predictor of mouse exposure dose, we performed multiple linear regression analysis at time points 0.25, 0.5,1,2 with SAA1 concentration and total peripheral blood lymphocytes as independent variables and radiation dose as dependent variables. The results show (Table 1) that the radiation dose in TBI mice can be measured for serum SAA1 alone within 2 days after exposureThe concentration, especially at day 0.5 after irradiation, had a correlation coefficient of R2 ═ 0.802(n ═ 48, P < 0.001). Furthermore, the concentration of SAA1 and the total number of peripheral blood lymphocytes were used as independent variables, and a good radiation dose estimation effect was obtained, particularly at 0.5 and 2 days after irradiation, with correlation coefficients of R2-0.829 (n-48, P < 0.001) and R2-0.840 (n-48, P < 0.001), respectively. From the above results, it can be seen that SAA1 and the irradiation dose show good linear regression, for example, 12 hours after irradiation, if SAA1 is taken as independent variable, when the concentration of SAA1 in serum is 25 μ g/ml, the irradiation dose is 4.389Gy can be calculated by formula; when SAA1 and lymphocyte number are used as independent variables for analysis, the serum SAA1 concentration is 25 μ g/ml, and lymphocyte number is 1 × 10⁹When the dosage is/L, the radiation dose is 4.845Gy by the formula.

TABLE 1 multiple Linear regression analysis between serum SAA1 concentration and lymphocyte count and irradiation dose

Note: n is the number of animals for which the regression equation was established.

In addition to radiation dose, it is also important to classify patients according to dose range. SAA1 thresholds were determined within 2 days post-irradiation using a subject-operator curve (ROC) to establish sensitivity, specificity and accuracy (table 2). When the irradiated individuals are irradiated for 6 hours, 2.155 mu g/ml can be used as a cut-off value to classify the irradiated individuals into two types of irradiation below 4Gy and irradiation above 8Gy, and the accuracy rate is 16/17, namely 16 of 17 individuals can be correctly classified; when the irradiated individuals are 12 hours after being irradiated, 3.26 mu g/ml can be used as a cut-off value to divide the irradiated individuals into two types of non-irradiated/irradiated 1Gy and irradiated with more than 2Gy, and the accuracy rate is 31/31, namely all 31 individuals can be correctly classified; when the irradiated individuals are irradiated for 1 day, 0.72 mu g/ml can be used as a cut-off value to divide the irradiated individuals into two types of non-irradiated/irradiated 1Gy and irradiated with more than 2Gy, and the accuracy rate is 32/33, namely 32 of 33 individuals can be correctly classified; when the number of irradiated individuals is 2 days after irradiation, 4.16 mug/ml can be used as a cut-off value to classify the irradiated individuals into two types of irradiation below 4Gy and irradiation above 8Gy, and the accuracy rate is 16/17, namely 16 of 17 individuals can be correctly classified.

TABLE 2 Critical values for SAA1 to differentiate the range of doses irradiated within 2 days after irradiation

Correlation of secondary increase in SAA1 with radiation lethality

It can be seen from g in fig. 2 and h in fig. 2 that the number of mice showing a second increase in SAA1 was about the same as the number of deaths (a in fig. 1) in the model group mice at

days

5 and 7 after the mice were irradiated with 8Gy and 12Gy systemically, which is also a crucial finding of the present invention, and the second increase in SAA1 is a predictor of radiation-induced deaths.

To assess whether a secondary increase in SAA1 is a useful indicator for predicting radiation-induced death, the present invention collected dynamic serum samples from 20 mice in a 10Gy group (5 times, 4 days prior to exposure and 1,3,5 and 7 days post exposure). The dynamic SAA1 concentrations and corresponding survival times of mice after 10Gy irradiation are listed in table 3, where each row represents the change in SAA1 concentration and corresponding survival time of consecutive blood samples from a single animal. A total of 92 unirradiated mice were used to calculate the mean and standard deviation of the SAA1 concentration (table 4), and a cut-off of 1.15 μ g/ml (mean +2 × standard deviation) was used to predict subsequent lethality within 30 days post-irradiation. On day 5, the concentration of SAA1 was higher than 1.15. mu.g/ml in 8 out of 17 dead and 3 live mice, the sensitivity was 8/17, the specificity was 2/3, and the accuracy was 8/9. In addition, on day 7, the concentration of SAA1 was higher than 1.15. mu.g/ml in 13 out of 16 dead mice and 1 out of 3 live mice with a sensitivity of 13/16, specificity of 2/3 and accuracy of 13/14.

TABLE 3.10 dynamic SAA1 concentration and corresponding time to death within 30 days after Gy irradiation

TABLE 4.92 Normal mice SAA1 concentration

After determining the correlation between the secondary increase in mouse SAA1 after irradiation and radiation lethality, we evaluated the effect of amifostine, a radioprotectant, on the secondary increase in mouse SAA1 after lethal dose irradiation to explore the effect of the secondary increase in mouse SAA1 after irradiation.

The experimental results showed that mice exposed to 10Gy began to die at day 7, and only 3 out of 20 mice survived to day 30, whereas in the amifostine-protected group (for 8 mice) administered prior to 10Gy irradiation, only 2 mice died at day 20 after irradiation, and the remaining 6 mice were all alive up to day 30. The above results further support that a secondary increase in mouse serum SAA1 following irradiation can be used as an indicator for predicting radiation lethality.

Therefore, in clinical applications, continuous monitoring of patient serum SAA1 concentration is of paramount importance for assessing prognosis. In addition, amifostine, a radioprotectant that reduces radiation damage, injected before 10Gy exposure inhibited the secondary increase in SAA1, while having no effect on the first increase in SAA1 after exposure. This result further confirms that a secondary increase in SAA1 is a useful indicator of survival after irradiation.

Example 2 detection of an increase in SAA1 following radiation therapy in patients with nasopharyngeal carcinoma.

After demonstrating that SAA1 can serve as a biomarker for mouse irradiation models, the present invention also examined whether SAA1 can serve as a biomarker for human irradiation models. Due to the limitation of ethical and moral level, the nasopharyngeal carcinoma patient receiving radiotherapy is used as a human irradiation model, and the serum SAA1 concentration of the tumor patient before and after radiotherapy is detected.

Patient and serum sample collection: serum samples of nasopharyngeal carcinoma patients signed with informed consent were collected in 17 southern hospitals in China. All specimens were from the clinical research initiative at southern medical university, as provided by the high-grade university construction Foundation (LC2016 PY015) at the education office of Guangdong province, and the https:// www.clinicaltrials.gov/_ research protocol was approved by the ethical Committee/institution (accession number: NFEC-2018-. The collimated beam of light using a 6MV linear accelerator encompasses the nasopharynx and its adjacent regions such as the posterior nasal fossa, part of the paranasal sinuses, the parapharyngeal space and the skull base. The total naso-and cervical doses were 70Gy and 63Gy, respectively. Blood specimens are collected 3-7 days before radiotherapy and 1-4 days after radiotherapy. The serum was then separated by centrifugation at 3000 rpm for 10 minutes at 25 ℃ and stored at-80 ℃ until use.

ELISA: SAA1 was prepared using an enzyme-linked immunosorbent assay (ELISA) kit as described in the manual.

Table 5 shows the clinical parameters and corresponding SAA1 concentrations for all 17 nasopharyngeal carcinoma patients considered in this study. As shown in a in FIG. 5, the serum concentration of SAA1 in each patient was elevated compared to that before irradiation, and the median post-radiotherapy concentration (44.63. mu.g/ml) was significantly higher than that before radiotherapy (0.54. mu.g/ml). FIG. 5 b shows the ROC curve for SAA1 as a biomarker for radiotherapy in nasopharyngeal carcinoma patients with an area under the curve (AUC) of 0.941. + -. 0.05, a cutoff of 9.34. mu.g/ml, a sensitivity of 18/18, a specificity of 16/18 and an accuracy of 18/20.

Table 5: clinical information of 17 nasopharyngeal carcinoma patients and SAA1 concentration

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Nucleotide amino acid sequence table

<110> military medical research institute of military science institute of people's liberation force of China

<120> model for predicting lethality of protein SAA1 in acute reaction phase after construction of ionizing radiation or preparation kit and reagent

In (1)

<130>

<160>6

<170>PatentIn version 3.5

<210>1

<211>369

<212>DNA

<213> CDS sequence of mouse SAA1

<400>1

atgaagctac tcaccagcct ggtcttctgc tccctgctcc tgggagtctg ccatggaggg 60

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gacatgaagg aagctaactg gaaaaactca gacaaatact tccatgctcg ggggaactat 180

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gaggcctttc aggaattctt cggcagagga catgaggaca ccattgctga ccaggaagcc 300

aacagacatg gccgcagtgg caaagacccc aattactaca gacctcctgg actgcctgac 360

aaatactga 369

<210>2

<211>122

<212>PRT

<213> mouse SAA1 amino acid sequence

<400>2

Met Lys Leu Leu Thr Ser Leu Val Phe Cys Ser Leu Leu Leu Gly Val

1 5 10 15

Cys His Gly Gly Phe Phe Ser Phe Val His Glu Ala Phe Gln Gly Ala

20 25 30

Gly Asp Met Trp Arg Ala Tyr Thr Asp Met Lys Glu Ala Asn Trp Lys

35 40 45

Asn Ser Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Gln

50 55 60

Arg Gly Pro Gly Gly Val Trp Ala Ala Glu Lys Ile Ser Asp Gly Arg

65 70 75 80

Glu Ala Phe Gln Glu Phe Phe Gly Arg Gly His Glu Asp Thr Ile Ala

85 90 95

Asp Gln Glu Ala Asn Arg His Gly Arg Ser Gly Lys Asp Pro Asn Tyr

100 105 110

Tyr Arg Pro Pro Gly Leu Pro Asp Lys Tyr

115 120

<210>3

<211>369

<212>DNA

<213> human SAA1 CDS sequence

<400>3

atgaagcttc tcacgggcct ggttttctgc tccttggtcc tgggtgtcag cagccgaagc 60

ttcttttcgt tccttggcga ggcttttgat ggggctcggg acatgtggag agcctactct 120

gacatgagag aagccaatta catcggctca gacaaatact tccatgctcg ggggaactat 180

gatgctgcca aaaggggacc tgggggtgcc tgggctgcag aagtgatcag cgatgccaga 240

gagaatatcc agagattctt tggccatggt gcggaggact cgctggctga tcaggctgcc 300

aatgaatggg gcaggagtgg caaagacccc aatcacttcc gacctgctgg cctgcctgag 360

aaatactga 369

<210>4

<211>122

<212>PRT

<213> human SAA1 amino acid sequence

<400>4

Met Lys Leu Leu Thr Gly Leu Val Phe Cys Ser Leu Val Leu Gly Val

1 5 10 15

Ser Ser Arg Ser Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala

20 25 30

Arg Asp Met Trp Arg Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr Ile

35 40 45

Gly Ser Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Lys

50 55 60

Arg Gly Pro Gly Gly Ala Trp Ala Ala Glu Val Ile Ser Asp Ala Arg

65 70 75 80

Glu Asn Ile Gln Arg Phe Phe Gly His Gly Ala Glu Asp Ser Leu Ala

85 90 95

Asp Gln Ala Ala Asn Glu Trp Gly Arg Ser Gly Lys Asp Pro Asn His

100 105 110

Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr

115 120

<210>5

<211>17

<212>DNA

<213>16S rRNA forward primer

<400>5

aacgcgaaga accttac 17

<210>6

<211>17

<212>DNA

<213>16S rRNA reverse primer

<400>6

cggtgtgtac aagaccc 17

Claims

1. Application of acute response phase protein SAA1 in construction of a post-ionizing radiation lethality prediction model.

2. The use of claim 1, wherein the dependent variable of the prediction model is a lethality index, and the lethality index is a secondary increase of the content of the protein SAA1 in the acute response stage in vivo after the subject to be tested receives ionizing radiation.

3. Use according to claim 1, wherein the ionizing radiation has an intensity of 8Gy or more.

4. Use of the acute phase protein SAA1 in the manufacture of a kit or reagent for assessing or predicting lethality following ionizing radiation.

5. The use of claim 4, wherein the secondary increase of the content of the acute response phase protein SAA1 in the test subject after irradiation is used as the target detection index of the kit.

6. Use according to claim 4, wherein the ionizing radiation has an intensity of 8Gy or more.

7. Application of acute reactive phase protein SAA1 in preparation of kit or reagent for evaluating efficacy and/or prognosis of ionizing radiation medicine.

8. Use according to claim 7, wherein the ionizing radiation has an intensity of 8Gy or more.

9. Application of acute response phase protein SAA1 in preparation of kit or reagent for detecting irradiated dose or distinguishing irradiated dose range after ionizing radiation.

10. The application of the combination of the acute response phase protein SAA1 and the number of peripheral blood lymphocytes in the preparation of a kit or a reagent for detecting the irradiated dose or distinguishing the irradiated dose range after ionizing radiation.