CN110890133B - Application method of acute reaction period protein SAA1 in constructing model for predicting lethality after ionizing radiation or preparing kit and reagent - Google Patents

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 the individual to be tested receives 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 the SAA1 and the number of the peripheral blood lymphocytes can be combined to detect the irradiated dose of the irradiated individual or distinguish the irradiated dose range, and can be used for preparing a related detection kit or reagent. The invention is beneficial to the evaluation and detection of radiation damage.

Description

Application method of acute reaction phase protein SAA1 in constructing model for predicting lethality after ionizing radiation or preparing kit and reagent

Technical Field

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

Background

Conditions such as nuclear terrorist attacks, nuclear power plant 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 exposed and whether radiation damage to the exposed person is fatal!

SAA1 is an acute phase protein that can rise about ten-fold in local or mild inflammation and hundreds-fold in the presence of severe infections in the body. SAA1 is synthesized primarily by the liver, 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 human tissues other than liver, such as breast tissue, colon, esophagus, kidney, large intestine, pituitary and spleen. The SAA1 protein 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, immunomodulation 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 irradiated dose, it can be determined whether the exposure dose of the patient requires carefully deployed medical resources and care.

However, because the individual damage degree varies greatly due to individual differences, it is only the final goal to directly evaluate the radiation damage degree. The identification of severely impaired individuals exposed to the same dose of radiation remains a challenge given the estimation of the radiation dose. Patent application CN 110308285A discloses that the combination of acute response phase protein SAA and IL-1 α as a combined marker can be used for evaluating the irradiation dose of the test individuals, but the combined marker is difficult to be used for predicting 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 predicting or evaluating the radiation lethality of an individual to be detected, 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 body in the acute reaction period after the individual to be detected receives the ionizing radiation.

Further defined, the secondary increase refers to the decrease after the content of the protein SAA1 in the body in the acute response period is increased for the first time and then is increased rapidly again after the subject to be tested receives the 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 a reagent for evaluating or predicting lethality after ionizing radiation.

Further defined, the secondary increase of the content of the acute reaction period protein SAA1 in the individuals to be detected after radiation is used as a target detection index of the kit.

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

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

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 reaction phase protein SAA1 in preparing a kit or 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 prophylaxis 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 is the target protein for evaluation.

Further defined, the secondary increase of the content of the acute reaction period protein SAA1 in the individuals to be detected after radiation is used as a target detection index of the kit.

Further defined, the secondary increase refers to the decrease after the increase of the content of the protein SAA1 in the body in the acute reaction period after the subject to be tested receives the ionizing radiation, and then the secondary increase is rapidly increased again.

In order to solve the problem of how to detect the irradiated dose of an 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 period protein SAA1 and the number of peripheral blood lymphocytes in 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 result shows that the serum SAA1 in the mice of each dose irradiation group is increased to different degrees within 6 hours, reaches a peak value within 12 hours, is reduced on day 3, the serum SAA1 level is increased for the second time from day 5 to day 7, the second increase is related to the subsequent death condition of the mice, the SAA1 increase is also detected in the human serum of nasopharyngeal carcinoma patients after radiotherapy, the experiment proves the feasibility of the SAA1 alone serving as a biological marker in human radiation exposure, and the second increase of the SAA1 after the individuals to be detected receive radiation can be used as an index for predicting radiation lethality, so that the experiment can be used for constructing a model for predicting the lethality after ionizing radiation and predicting the radiation lethality. The invention also proves that the radiation dose of the irradiated individual can be estimated by the SAA1 or the SAA1 combined with the number of the peripheral blood lymphocytes, and the dose range can be classified, thereby being beneficial to identifying the damaged individual.

Drawings

FIG. 1. 30-day survival analysis, body weight analysis and peripheral blood cell analysis of mice irradiated systemically with different doses, wherein a in FIG. 1 is the 30-day survival of 0,1,2,4, 8 and 12Gy irradiated female C57BL/6J mice; b in FIG. 1 is the body weight of female C57BL/6J mice irradiated at different times with 0,1,2,4, 8 and 12 Gy; c, d, e, f in fig. 1 are the changes in the number of lymphocytes, erythrocytes, platelets and leukocytes 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, wherein a in FIG. 2 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, C, d, e, f, g, h in fig. 2 are the SAA1 concentrations as a function of irradiation dose at 0.25,0.5,1,2,3,5 and 7 days after 0,1,2,4, 8 and 12Gy irradiation of female C57BL/6J mice, respectively, with each black dot representing one animal and each group n =8;

FIG. 3 analysis of SAA1 concentration in serum of mice after irradiation and systemic infection parameters, wherein a in FIG. 3 is day 0,1,5 and 7 after 8Gy systemic irradiation, SAA1 and LPS in serum of 31 mice (7 mice on day 7, 8 mice in each group on day 0,1 and 5, no.1-No.8 of a in FIG. 3), and 16S rRNA expression in liver; b in FIG. 3 is 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, and No.1-No.3 of b in FIG. 3) on days 0, 0.125, 0.5,1,2,3,5, and 7 after 12Gy total body irradiation;

FIG. 4 shows the serum SAA1 profile of a partially irradiated mouse, and a in FIG. 4 shows the irradiation of a C57BL/6J mouse, in which the liver was shielded in PBI-1 and PBI-3 and exposed in PBI-2 and PBI-4 groups; b in FIG. 4 is the concentration of SAA1 in serum of the control, whole body irradiation and PBI groups at 12 hours after 8Gy irradiation, and c in FIG. 4 is the mRNA expression level of SAA1 in liver of the control, whole body irradiation and PBI groups; n =6 per group, compared to the control group, ^* P＜0.05， ^** P＜0.01， ^*** p < 0.001 and ^**** P＜0.0001；

FIG. 5. Effect of radiotherapy on serum SAA1 of nasopharyngeal carcinoma patients, a scatter plot shown in FIG. 5 shows SAA1 concentration before and after radiotherapy (P < 0.0001) in 17 cases of nasopharyngeal carcinoma; FIG. 5 b is a ROC curve of SAA1 as a biomarker for predicting radiation exposure of 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 are performed according to corresponding instructions.

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

The nucleotide sequence of the acute reaction phase protein SAA1 and the murine SAA1 is shown in 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) kits are used for detecting SAA1 and Procalcitonin (PCT), wherein (mouse SAA1 ELISA kit product information: EK1190, BOSTER, wuhan, china; human SAA1 ELISA kit product information: EK1544, BOSTER, wuhan, china; PCT ELISA kit product information: E1071M, cuxabo, wuhan, china, and the operation method is as per the instruction.

The product information of limulus reagent for determining lipopolysaccharide includes 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 time and dose response studies, 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 12Gy.

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 of the dynamic monitoring of the SAA1 concentration, the mouse is randomly bound with the earrings marked with random numbers, the mouse is raised for 5 days after the ear rings are bound until the normal state is recovered, the mouse in the 10Gy irradiation group is placed in an organic glass transparent box and receives the mice with the dosage rate of 73.02cGy/min ⁶⁰ The mice in each group were irradiated with Co-source gamma rays at a total dose of 10Gy, venous blood was collected from the tail vein at a dose of about 40. Mu.L 4 days before irradiation and 1,3,5, and 7 days after irradiation, respectively, and survival of the mice in each group was observed within 30 days after irradiation.

Local irradiation (PBI) mice were intraperitoneally injected with 10mg/kg sodium pentobarbital, anesthetized, placed in a plexiglass box, placed in 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 thermosetting 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 of DNA and quantitative PCR analysis

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, used for quantitative PCR on a BIORAD CFX96 instrument using iTaq Universal SYBR Green Supermix.

Statistical analysis was performed using IBM SPSS 21. The data of normal distribution is subjected to t test to evaluate the difference between two groups, and the data of non-normal distribution adopts non-parametric test. Serum SAA1 levels were statistically analyzed by multiple linear regression analysis as a predictor of irradiated dose feasibility after whole body irradiation. A nonparametric Spman rank correlation method is adopted, binary correlation analysis is carried out on the SAA1, the LPS and the 16S rRNA of the 8Gy irradiated mouse, and correlation analysis is carried out on the SAA1 and the PCT of the 12Gy irradiated mouse. Determining a predictive cutoff value from a Receiver Operating Characteristic (ROC) curve; the survival distribution is determined by Log-rank test; the patient before and after radiotherapy was tested for SAA1 concentration with Mann Whitney u.

The results were as follows:

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

Mice were irradiated at different doses of 0,1,2,4, 8 and 12Gy, and observed for changes in survival rate, body weight and peripheral blood cell count after irradiation. As shown in a of 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 of serum SAA1 concentration of C57BL/6J mice caused by whole body irradiation

To evaluate the time and dose effects of serum SAA1 concentration after systemic irradiation of mice, serum SAA1 concentrations were measured by ELISA method at 0.25,0.5,1,2,3,5 and 7 days after systemic irradiation of C57BL/6J mice with 1,2,4, 8 and 12Gy. In fig. 2, a shows that the concentration of SAA1 in each dose group starts to increase at 6 hours after the irradiation, reaches a peak at 12 hours, and then substantially returns to the initial level on day 3. In FIG. 2, b, c, d, e, f, g and h show the SAA1 concentration at 0.25,0.5,1,2,3,5 and 7 days after each dose. On day 5 post-irradiation, 2 of 8 mice in the 8Gy group showed a large re-increase in SAA1 (60.66. Mu.g/ml and 113.26. Mu.g/ml, the same units are used below), and 6 of 8 mice in the 12Gy group showed a re-increase in SAA1 (109.62, 301.87,365.43,424.15,332.16, 284.66. Mu.g/ml). On day 7 after the photograph, 4 of 8 mice in the 8Gy group had a large increase in SAA1 again (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. Serum SAA1 elevation 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 the bacteria, present in the genome 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. Using the parameters after 7 days of 8Gy irradiation as examples, the SAA1 concentrations of 7 mice were 156.79, 0.12, 18.92, 0.51, 106.12,170.22 and 107.31. Mu.g/ml, the LPS levels were 0.97,0.03,0.18,1.56,0.03,0.06 and 0.98EU/ml, and the ploidy change of 16S rRNA in the liver was: 1.14,4.03,1.73,1.28,2.39,3.44 and 3.39 times. In addition to measuring direct bacterial infection indicators (LPS and 16S rRNA), 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 serum PCT concentration (a marker of bacterial infection) levels on days 0, 0.25,0.5,1,2,3,5, 7 after irradiation, and analyzed their correlation with SAA1 expression levels. 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 infection. LPS as an exogenous Toll-like receptor ligand inducing SAA1 and PCT is a biomarker for bacteremia. The 16S rRNA gene is highly conserved in bacteria and is a standard method for identifying and classifying prokaryotes. According to the invention, by measuring SAA1, PCT and LPS in the same serum and evaluating 16S rRNA expression in the liver of the same mouse, the results of bivariate correlation analysis show that the serum SAA1 concentration of the 8Gy whole body irradiated mouse has no correlation with LPS activity and liver 16S rRNA expression quantity. There was no correlation between serum SAA1 and PCT concentration in 12Gy whole body irradiated mice, i.e., SAA1 was increased without being correlated 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 changes in SAA1 in mice irradiated under liver-shielding conditions are not clear. To investigate the varying levels of SAA1 in liver-shielded conditions, animals were irradiated by 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), exposing the posterior flank and hind limbs (PBI-3), as shown in fig. 4 a, the only difference between PBI-1 and PBI-2 was 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 mouse serum SAA1 and liver SAA1 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 systemic dose threshold at 0.5 days post irradiation was also applicable to the local irradiation, 3.26 μ g/ml calculation control and PBI groups (PBI-1, PBI-2, PBI-3, PBI-4) were selected, resulting in a sensitivity of 22/24, specificity of 24/24, and accuracy of 24/24. Thus, it can be demonstrated that the SAA1 concentration threshold of 0.5 days after whole 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 fraction exposure (PBI-2 and PBI-4) and shielded liver fraction exposure (PBI-1 and PBI-3) to obtain a cut-off of 11.46. Mu.g/ml, sensitivity 12/12, specificity 11/12, and precision 12/13.

SAA1 prediction of whole body irradiation dose and differentiation of irradiation dose range in mice

To investigate whether serum SAA1 levels can be used as a predictor of mouse exposure dose, we performed multiple linear regression analysis at time points of 0.25,0.5, and 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 concentration alone within 2 days after exposure, especially at 0.5 days after irradiation, with a correlation coefficient of R2=0.802 (n =48, p < 0.001). Furthermore, taking the concentration of SAA1 and the total number of peripheral blood lymphocytes as independent variables, better radiation dose estimation results were obtained, especially 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 irradiation dose exhibit good linear regression, for example, 12 hours after irradiation, if SAA1 alone is used as an independent variable, when the concentration of SAA1 in serum is 25 μ g/ml, the irradiation dose can be calculated as 4.389Gy by formula; when SAA1 and lymphocyte count are used as independent variables for analysis, when serum SAA1 concentration is 25 μ g/ml, lymphocyte count is 1 × 10 ⁹ at/L, the dose of 4.845Gy can be calculated 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 data was used to establish the regression equation.

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 divide 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 irradiation is carried out for 2 days, 4.16 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.

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

Correlation between secondary increase of SAA1 and radiation lethality

As can be seen from g in FIG. 2 and h in FIG. 2, the number of mice showing the second increase of SAA1 on days 5 and 7 after the mice were irradiated with 8Gy and 12Gy systemically was approximately the same as the number of deaths (a in FIG. 1) in the model group mice, which is also a crucial finding of the present invention, and the second increase of 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 collects dynamic serum samples from 20 mice in a 10Gy group (5 times, 4 days before exposure and 1,3,5 and 7 days after exposure). The dynamic SAA1 concentration and corresponding survival time of mice after 10Gy irradiation are listed in Table 3, where each row represents the change in SAA1 concentration and corresponding survival time for successive blood samples from a single animal. A total of 92 unirradiated mice were used to calculate the mean and standard deviation of 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, SAA1 concentration was higher than 1.15. Mu.g/ml in 8 out of 17 dead mice and 1 out of 3 live mice, with a sensitivity of 8/17, specificity of 2/3 and accuracy of 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, a specificity of 2/3 and an accuracy of 13/14.

TABLE 3.10 dynamic SAA1 concentration and corresponding death time 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 after 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 SAA1 elevation following radiation therapy in nasopharyngeal carcinoma patients.

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 darcinoma 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: 17 serum samples of nasopharyngeal carcinoma patients signed with informed consent were collected in southern hospitals in China. All specimens were from the clinical research initiative at southern medical university, provided by the high-grade university construction foundation (LC 2016 PY 015) at the education office of Guangdong province, https:// www.clinicaltirials.gov/research protocol was approved by the ethical committee/agency (accession number: NFEC-2018-013). The collimated beam of light using a 6MV linear accelerator encompasses the nasopharynx and its adjacent regions such as the posterior nasal fossa, parts of the sinuses, parapharyngeal space and the skull base. The total naso-and cervical doses were 70Gy and 63Gy, respectively. Blood samples 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 performed using enzyme-linked immunosorbent assay (ELISA) kit protocol 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 SAA1 concentration of 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 of 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 and SAA1 concentration of 17 nasopharyngeal carcinoma patients

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 the people's liberation army 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> mouse SAA1 CDS sequence

<400> 1

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gatgctgctc aaaggggtcc cgggggagtc tgggctgctg agaaaatcag tgatggaaga 240

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 (3)

1. An application method of acute reactive phase protein SAA1 in constructing a post-ionizing radiation lethality prediction model is characterized in that a dependent variable of the post-ionizing radiation lethality prediction model is a lethal index, and the lethal index is a secondary increase of the content of the in-vivo acute reactive phase protein SAA1 after an individual to be detected receives ionizing radiation; the secondary increase refers to that after the individual to be detected receives ionizing radiation, the content of the protein SAA1 in the acute reaction phase in vivo is decreased after being increased for the first time, and then is rapidly increased again; the intensity of the ionizing radiation is 8Gy or more.

2. An application method of the acute reaction phase protein SAA1 in preparing a kit or a reagent for evaluating or predicting lethality after ionizing radiation is characterized in that the secondary increase of the content of the acute reaction phase protein SAA1 after an individual to be detected is irradiated is used as a target detection index of the kit or the reagent; the SAA1 can be used as a detection target alone to obtain an evaluation or prediction result; the secondary increase refers to that after an individual to be detected receives ionizing radiation, the content of the protein SAA1 in the acute reaction phase in vivo is decreased after being increased for the first time, and then is rapidly increased again; the intensity of the ionizing radiation is 8Gy or more.

3. An application method of the acute reaction phase protein SAA1 in preparing a kit or a reagent for evaluating the drug effect and/or prognosis of an ionizing radiation drug is characterized in that the secondary increase of the content of the acute reaction phase protein SAA1 after an individual to be detected is irradiated is used as an individual target detection index of the kit or the reagent; the secondary increase refers to that after an individual to be detected receives ionizing radiation, the content of the protein SAA1 in the acute reaction phase in vivo is decreased after being increased for the first time, and then is rapidly increased again; the intensity of the ionizing radiation is 8Gy or more.

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