CN110221337B - A method for evaluating biological damage of uranium ore dust by internal irradiation using α-1 antiprotease - Google Patents

Abstract

The invention relates to a method for evaluating irradiation biological damage in uranium ore dust by utilizing alpha-1 protease resistance. According to the dose-effect relationship between the amount of uranium ore dust suspension subjected to tracheal instillation by WISTAR rats and the up-regulation rate of the relative expression amount of alpha-1 protease resistance in lung tissues, the biological damage caused by the irradiation in the uranium ore dust is evaluated through the up-regulation rate of the relative expression amount of alpha-1 protease resistance in the lung tissues of the WISTAR rats by utilizing the characteristic that alpha-1 protease resistance in the lung tissues of the WISTAR rats is very sensitive to biological damage caused by the irradiation in the uranium ore dust. The method has the advantages of high sensitivity, strong specificity, good reliability and the like on biological damage caused by internal irradiation of uranium ore dust.

Description

Translated fromChinese

一种利用α-1抗蛋白酶评价铀矿粉尘内照射生物损伤的方法A method for evaluating biological damage of uranium ore dust by internal irradiation using α-1 antiprotease

技术领域technical field

本发明属于铀矿粉尘内照射生物损伤评价技术领域，利用分子标记物进行铀矿粉尘内照射生物损伤评价的方法，具体是利用WISTAR大鼠肺组织中α-1抗蛋白酶的相对表达量上调率来评价铀矿粉尘内照射生物损伤的方法。The invention belongs to the technical field of internal irradiation biological damage evaluation of uranium ore dust, and uses molecular markers to evaluate the biological damage of uranium ore dust internal irradiation. A method for evaluating biological damage from radiation in uranium ore dust.

背景技术Background technique

在铀矿勘探、开采、水冶和尾矿处置过程中都会产生一定数量的铀矿粉尘污染，这些铀矿粉尘中含有铀、钍、镭等放射性核素，一旦被人体吸进体内，就会长时间滞留于肺气管、支气管和淋巴结内，构成潜在的长期内照射危害，产生生物损伤。铀矿尘的电离辐射主要来自于氡及其子体(²¹⁸Po、²¹⁴Pb、²¹⁴Bi 和²¹⁴Po)衰变产生的 α、β射线，由于吸入的粉尘产生的放射性剂量低，短时间内很难表现出生物效应，因而难以进行检测和生物损伤评估。因此，筛选出对铀矿粉尘内照射造成的生物损伤检测速度快、敏感性高、特异性强、可靠性好的生物标志物，对具有十分重要的意义。A certain amount of uranium dust pollution will be generated in the process of uranium exploration, mining, hydrometallurgy and tailings disposal. These uranium dusts contain radionuclides such as uranium, thorium and radium. Long-term retention in the pulmonary trachea, bronchi and lymph nodes constitutes a potential long-term internal radiation hazard, resulting in biological damage. The ionizing radiation of uranium dust mainly comes from the alpha and beta rays produced by the decay of radon and its daughters (²¹⁸ Po,²¹⁴ Pb,²¹⁴ Bi and²¹⁴ Po). Demonstrates biological effects, making detection and assessment of biological damage difficult. Therefore, it is of great significance to screen out biomarkers with high detection speed, high sensitivity, specificity and reliability for biological damage caused by internal irradiation of uranium ore dust.

生物体吸入铀矿粉尘后，可能通过改变蛋白质的表达水平及其翻译后修饰状态来响应电离辐射。iTRAQ 技术是新兴的高通量蛋白质组学方法，通过特异性标记多肽的氨基基团，然后进行串联质谱分析，可同时比较4种或8种不同样品中蛋白质的相对含量或绝对含量。因此，可以通过iTRAQ 技术筛选组织样品中差异表达的蛋白质来确定与铀矿粉尘内照射造成的生物损伤相关的生物标志物，进而采用筛选的生物标志物对铀矿粉尘内照射造成生物损伤进行评价。After inhalation of uranium ore dust, organisms may respond to ionizing radiation by altering protein expression levels and their post-translational modification states. iTRAQ technology is an emerging high-throughput proteomics method that can simultaneously compare the relative or absolute content of proteins in 4 or 8 different samples by specifically labeling amino groups of polypeptides and then performing tandem mass spectrometry analysis. Therefore, iTRAQ technology can be used to screen differentially expressed proteins in tissue samples to determine biomarkers related to biological damage caused by internal irradiation of uranium ore dust, and then use the screened biomarkers to evaluate biological damage caused by internal irradiation of uranium ore dust. .

发明内容SUMMARY OF THE INVENTION

针对上述情况，本发明提供一种利用WISTAR大鼠肺组织中α-1抗蛋白酶(Alpha-1-antiproteinase，A1AT) 的相对表达量上调率来评价铀矿粉尘内照射造成的生物损伤的方法。本发明利用WISTAR大鼠肺组织中α-1抗蛋白酶对铀矿粉尘内照射造成的生物损伤非常敏感的特性，根据WISTAR大鼠接受气管滴注的铀矿粉尘混悬液（铀矿粉尘与生理盐水按照一定比例配置）的量与肺组织中α-1抗蛋白酶相对表达量的上调率的剂量-效应关系，通过WISTAR大鼠肺部组织中α-1抗蛋白酶的相对表达量的上调率，评估铀矿粉尘内照射造成的生物损伤。其原理是， 发明人通过iTRAQ 技术筛选发现接受气管滴注的铀矿混悬液后WISTAR大鼠肺部组织中的α-1抗蛋白酶的相对表达量对铀矿粉尘非常敏感，且存在一定的剂量-效应关系。α-1抗蛋白酶是血浆浓度响应炎症而变化的蛋白质，参与氧化应激，炎症反应和细胞凋亡。在一定的内照射剂量范围内，机体受到的内辐照损伤越大，α-1抗蛋白酶的相对表达量就越高，α-1抗蛋白酶蛋白表达的高低直接与铀矿粉尘内照射造成的生物损伤程度密切相关，该方法具有对铀矿粉尘内照射造成的生物损伤敏感性高、特异性强、可靠性好等多重优点。In view of the above situation, the present invention provides a method for evaluating biological damage caused by internal irradiation of uranium ore dust by using the relative expression up-regulation rate of alpha-1-antiproteinase (A1AT) in lung tissue of WISTAR rats. The present invention utilizes the characteristic that α-1 anti-protease in the lung tissue of WISTAR rats is very sensitive to biological damage caused by internal irradiation of uranium ore dust, according to the uranium ore dust suspension (uranium ore dust and physiological The dose-response relationship between the amount of saline (prepared in a certain proportion) and the up-regulation rate of the relative expression of α-1 anti-protease in lung tissue, through the up-regulation rate of the relative expression of α-1 anti-protease in the lung tissue of WISTAR rats, Evaluation of biological damage from internal exposure to uranium ore dust. The principle is that the inventors screened through iTRAQ technology and found that the relative expression of α-1 antiprotease in the lung tissue of WISTAR rats after receiving the uranium ore suspension by tracheal instillation is very sensitive to uranium ore dust, and there is a certain amount of uranium ore dust. Dose-response relationship. Alpha-1 antiproteases are proteins whose plasma concentrations vary in response to inflammation and are involved in oxidative stress, inflammatory responses and apoptosis. Within a certain range of internal radiation dose, the greater the internal radiation damage to the body, the higher the relative expression of α-1 anti-protease. The degree of biological damage is closely related, and this method has multiple advantages such as high sensitivity, specificity and reliability to the biological damage caused by internal irradiation of uranium ore dust.

具体步骤是：The specific steps are:

（1）建立铀矿混悬液气管滴注WISTAR大鼠模型；(1) Establish a rat model of uranium ore suspension tracheal instillation of WISTAR;

（2）采集肺组织；(2) Collect lung tissue;

（3）α-1抗蛋白酶相对表达量上调率的检测；(3) Detection of the up-regulation rate of the relative expression of α-1 anti-protease;

（4）内照射生物损伤综合评价。(4) Comprehensive evaluation of biological damage from internal irradiation.

其进一步的措施是：Its further steps are:

所述的建立铀矿混悬液气管滴注WISTAR大鼠模型的具体方法是：The specific method for establishing the WISTAR rat model of uranium ore suspension tracheal instillation is:

选择体重在180 - 220 g之间的WISTAR成年雄性大鼠，随机分4组，每组6只老鼠，对照组气管滴注生理盐水和二氧化硅的悬液（二氧化硅的粒径为200目），实验组分别气管滴注浓度为1.25、2.5和5.0 mg/ml的铀矿粉尘悬液（铀矿粉尘的粒径为200目），每次滴注0.2 ml，一周滴注3次，连续5周。WISTAR adult male rats weighing between 180 and 220 g were selected and randomly divided into 4 groups, with 6 rats in each group. The control group was instilled with saline and a suspension of silica (the particle size of silica was 200 In the experimental group, uranium ore dust suspension (the particle size of uranium ore dust is 200 mesh) with concentrations of 1.25, 2.5 and 5.0 mg/ml was instilled through the trachea, 0.2 ml each time, 3 times a week, 5 consecutive weeks.

所述的采集肺组织的具体方法是：The specific method for collecting lung tissue is:

用颈椎脱臼法处死大鼠，解剖后取肺组织，把各组织分成3等份，用生理盐水冲洗掉血水，除去水分，装到冻存管内，马上放入-80℃冰箱保存。The rats were killed by cervical dislocation. After dissection, lung tissue was taken, and each tissue was divided into 3 equal parts. The blood was rinsed with normal saline to remove the water, and it was placed in a cryopreservation tube and immediately stored in a -80 ℃ refrigerator.

所述的α-1抗蛋白酶相对表达量上调率的检测具体方法是：The specific method for detecting the up-regulation rate of the relative expression of α-1 antiprotease is:

将收集的接受过铀矿粉尘内照射WISTAR大鼠肺部组织进行蛋白提取、总蛋白浓度测定、蛋白质变性、凝胶电泳、转膜、免疫反应、化学发光反应、凝胶成像扫描分析、最后进行α-1抗蛋白酶的相对表达量上调率分析。The collected lung tissues of WISTAR rats that had been irradiated with uranium ore dust were subjected to protein extraction, total protein concentration determination, protein denaturation, gel electrophoresis, membrane transfer, immunoreaction, chemiluminescence reaction, gel imaging scanning analysis, and finally Analysis of the relative expression up-regulation of α-1 antiprotease.

所述的蛋白提取具体步骤如下：The specific steps of the protein extraction are as follows:

在研钵中倒入液氮预冷，迅速取出肺部组织样品，把组织切至均匀置于研钵中，马上加入液氮，进行研磨。在研钵中慢慢加入800 μl LB裂解液，裂解30 min，振荡1-2次，充分裂解。裂解结束后，收集1.5 mL的悬液到EP管中，然后在4℃下，转速12000 r/min，离心20min后，收集上清液。用树脂沉淀上清液中的蛋白，4℃，12000 r/min离心20 min，去掉上清，晾干，-80℃保存备用。在干燥后的蛋白质团块中加入200-250 μL L3裂解液，用枪头反复吹打直至蛋白质充分分散。超声助溶，4℃，12000 r/min，离心20 min，吸出上清液，转移至新的EP管中。Pour liquid nitrogen into the mortar to pre-cool, quickly take out the lung tissue samples, cut the tissue into a mortar, and immediately add liquid nitrogen for grinding. Slowly add 800 μl LB lysis solution to the mortar, lyse for 30 min, shake 1-2 times, and fully lyse. After the lysis, 1.5 mL of the suspension was collected into an EP tube, and then centrifuged at 12,000 r/min at 4 °C for 20 min, and the supernatant was collected. The protein in the supernatant was precipitated with resin, centrifuged at 12,000 r/min for 20 min at 4°C, the supernatant was removed, air-dried, and stored at -80°C for later use. Add 200-250 μL of L3 lysis solution to the dried protein clump, and pipet repeatedly with a pipette tip until the protein is fully dispersed. Ultrasonic aided dissolution, 4 °C, 12000 r/min, centrifugation for 20 min, aspirate the supernatant, and transfer to a new EP tube.

所述总蛋白浓度测定的具体步骤如下：The specific steps for the determination of the total protein concentration are as follows:

将0.2 mg/ml 的BSA 标准品溶液加稀释液，依次稀释成0、5、10、20、50、100、150和200 μg/ml 的标准品溶液。然后测定它们在562 nm下的吸光度值，绘制标准曲线。测定每个样品溶液在562 nm下的吸光度值后，再通过标准曲线计算出样品中蛋白质的浓度。Add the 0.2 mg/ml BSA standard solution to the diluent and dilute to 0, 5, 10, 20, 50, 100, 150 and 200 μg/ml standard solution. Then measure their absorbance values at 562 nm and draw a standard curve. After measuring the absorbance value of each sample solution at 562 nm, the protein concentration in the sample was calculated through the standard curve.

所述蛋白质变性的具体步骤如下：The specific steps of the protein denaturation are as follows:

将蛋白质溶液按照4:1的比例加入5×蛋白质上样缓冲液，沸水浴变性15 min，放入-20 ℃冰箱保存备用。The protein solution was added to 5× protein loading buffer at a ratio of 4:1, denatured in a boiling water bath for 15 min, and stored in a -20 °C refrigerator for later use.

所述凝胶电泳的具体步骤如下：The specific steps of the gel electrophoresis are as follows:

将30％丙烯酰胺、1.5 M TRIS－HCl、10％SDS、10% 过硫酸铵和TEMED按照一定比例配制成10%的分离胶和5%的浓缩胶；待分离胶凝固后，在电泳槽中加入电泳液，将变性后的蛋白质样品加入电泳孔中，进行电泳。将浓缩胶电压调至75 V，分离胶电压调至100 V，电泳至溴酚蓝刚跑至分离胶底部，即可终止电泳，进行转膜。30% acrylamide, 1.5 M TRIS-HCl, 10% SDS, 10% ammonium persulfate and TEMED were prepared into 10% separating gel and 5% stacking gel according to a certain proportion; The electrophoresis solution is added, and the denatured protein sample is added to the electrophoresis well for electrophoresis. Adjust the voltage of the stacking gel to 75 V and the voltage of the separating gel to 100 V, and electrophoresis until the bromophenol blue has just run to the bottom of the separating gel, then the electrophoresis can be terminated and the membrane can be transferred.

所述转膜的具体步骤如下：The specific steps of the film transfer are as follows:

先采用甲醇将PVDF膜进行活化，再在膜的底部放置5-6张7×9 cm的滤纸，再一同放置于转膜仪中，加入转膜液，300 mA恒流转膜30 min。转膜过程中，将转膜槽放置于冰水中降温。First, the PVDF membrane was activated with methanol, and then 5-6 pieces of 7 × 9 cm filter paper were placed at the bottom of the membrane, and then placed in the membrane transfer apparatus. During the transfer process, place the transfer tank in ice water to cool down.

所述免疫反应的具体步骤如下：The specific steps of the immune response are as follows:

将转好的膜用5%的脱脂牛奶（0.5% TBST稀释）封闭，在脱色摇床上混匀1 h。采用TBST溶解的5%脱脂牛奶对α-1抗蛋白酶一抗（Alah3al）进行1:1000浓度稀释，然后将其与封闭好的膜进行混匀，4 ℃下孵育过夜。用2% TBST对α-1抗蛋白酶一抗孵育过夜的膜进行清洗，室温下在脱色摇床上清洗3次，每次5 min。最后用TBST对二抗（羊抗兔）稀释8000倍，室温下孵育1.5 h后，在脱色摇床上清洗三次，每次10 min。The transferred membrane was blocked with 5% skim milk (diluted with 0.5% TBST) and mixed for 1 h on a destaining shaker. The α-1 anti-protease primary antibody (Alah3al) was diluted 1:1000 with 5% nonfat milk dissolved in TBST, then mixed with the blocked membrane, and incubated overnight at 4 °C. The membrane incubated overnight with α-1 anti-protease primary antibody was washed with 2% TBST, and washed three times at room temperature on a destaining shaker for 5 min each time. Finally, the secondary antibody (goat anti-rabbit) was diluted 8000 times with TBST, incubated at room temperature for 1.5 h, and washed three times on a destaining shaker for 10 min each time.

所述化学发光反应的具体步骤如下：The specific steps of the chemiluminescence reaction are as follows:

在暗室中将ECLA和ECLB两种试剂在离心管中等体积混合，将PVDF膜的蛋白面朝上，加入混合好的ECL溶液充分反应1-2 min后，放入凝胶成像系统进行扫描分析。Mix the two reagents, ECLA and ECLB, in a centrifuge tube in an equal volume in a dark room, put the protein side of the PVDF membrane up, add the mixed ECL solution and react for 1-2 minutes, then put it into the gel imaging system for scanning and analysis.

所述的凝胶成像扫描分析的具体步骤如下：The specific steps of the gel imaging scanning analysis are as follows:

采用凝胶成像系统对蛋白条带进行拍照，再采用Image J软件分析目标带的灰度值。通过对α-1抗蛋白酶蛋白灰度值扫描获得α-1抗蛋白酶蛋白表达量，对内参蛋白GAPDH蛋白灰度值扫描获得GAPDH蛋白表达量；将α-1抗蛋白酶蛋白表达量除以内参蛋白GAPDH蛋白表达量即获得α-1抗蛋白酶的相对表达量。The protein bands were photographed by a gel imaging system, and the gray values of the target bands were analyzed by Image J software. The expression of α-1 anti-protease protein was obtained by scanning the gray value of α-1 anti-protease protein, and the expression of GAPDH protein was obtained by scanning the gray value of internal reference protein GAPDH protein; the expression of α-1 anti-protease protein was divided by the internal reference protein. The GAPDH protein expression level is the relative expression level of α-1 antiprotease.

所述α-1抗蛋白酶的相对表达量上调率分析的具体步骤如下：The specific steps for the analysis of the relative expression up-regulation rate of the α-1 antiprotease are as follows:

α-1抗蛋白酶(A1AT)相对表达量上调率按公式（1）进行计算：The relative expression up-regulation rate of α-1 antiprotease (A1AT) was calculated according to formula (1):

（1）

(1)

所述内照射生物损伤综合评价的具体方法是：The specific method for the comprehensive evaluation of the internal irradiation biological damage is:

采用α-1抗蛋白酶的相对表达量上调率来评价铀矿粉尘内照射造成的生物损伤。当WISTAR大鼠肺部组织中的α-1抗蛋白酶的相对表达量上调率在30%以内时，表明低剂量伽马射线辐照对无生物损伤效应；当WISTAR大鼠肺部组织中的α-1抗蛋白酶的相对表达量上调率大于30%而小于100%时，此时的铀矿粉尘内照射造成的生物损伤等级为Ⅰ级；当WISTAR大鼠肺部组织中的α-1抗蛋白酶的相对表达量上调率大于100%而小于200%时，此时的铀矿粉尘内照射造成的生物损伤等级为Ⅱ级；当WISTAR大鼠肺部组织中的α-1抗蛋白酶的相对表达量上调率大于200%时，此时的铀矿粉尘内照射造成的生物损伤等级为Ⅲ级。当铀矿粉尘内照射造成的生物损伤等级达到Ⅰ级时，职业人员应加强辐照防护；当铀矿粉尘内照射造成的生物损伤等级达到Ⅱ级时，职业人员应及时休息一段时间；当铀矿粉尘内照射造成的生物损伤等级达到Ⅲ级时，职业人员应调离工作岗位，并接受适当治疗。The relative expression up-regulation rate of α-1 antiprotease was used to evaluate the biological damage caused by internal irradiation of uranium ore dust. When the relative expression of α-1 antiprotease in the lung tissue of WISTAR rats was up-regulated within 30%, it indicated that low-dose gamma irradiation had no biological damage effect; When the relative expression of -1 anti-protease increased by more than 30% but less than 100%, the biological damage level caused by internal irradiation of uranium ore dust was grade I; When the relative expression up-regulation rate of α-1 antiprotease in WISTAR rats was greater than 100% but less than 200%, the biological damage level caused by internal irradiation of uranium ore dust was grade II; when the relative expression level of α-1 antiprotease in the lung tissue of WISTAR rats When the upward adjustment rate is greater than 200%, the biological damage level caused by internal irradiation of uranium ore dust at this time is level III. When the biological damage level caused by internal irradiation of uranium ore dust reaches level I, professionals should strengthen radiation protection; when the biological damage level caused by internal irradiation of uranium ore dust reaches level II, professionals should take a break in time; When the biological damage level caused by the internal irradiation of mineral dust reaches level III, the professional personnel should be transferred from their jobs and receive appropriate treatment.

本专利发明是利用蛋白质组学全定量iTRAQ 技术分析差异表达的蛋白，鉴定α-1抗蛋白酶作为铀矿粉尘内照射造成的生物损伤的生物标志物，然后利用α-1抗蛋白酶的相对表达量与铀矿粉尘内照射造成的生物损伤之间的剂量-效应关系来评价铀矿粉尘内照射造成的生物损伤。相比于其他的生物标志物及评价方法，该方法具有对铀矿粉尘内照射造成的生物损伤敏感性高、特异性强、可靠性好等多重优点。The patented invention is to use the proteomics full quantitative iTRAQ technology to analyze differentially expressed proteins, identify α-1 anti-protease as a biomarker of biological damage caused by internal irradiation of uranium ore dust, and then use the relative expression of α-1 anti-protease. The dose-response relationship between the biological damage caused by internal irradiation of uranium ore dust was used to evaluate the biological damage caused by internal irradiation of uranium ore dust. Compared with other biomarkers and evaluation methods, this method has multiple advantages such as high sensitivity, specificity and reliability for biological damage caused by internal irradiation of uranium ore dust.

具体实施方式Detailed ways

实施例1Example 1

选择体重在180 - 220 g之间的WISTAR成年雄性大鼠，随机分4组，每组6只老鼠，对照组气管滴注生理盐水和二氧化硅的悬液（二氧化硅的粒径为200目），实验组分别气管滴注浓度为1.25 mg/ml的铀矿粉尘悬液（铀矿粉尘的粒径为200目），每次滴注0.2 ml，一周滴注3次，连续5周。用颈椎脱臼法处死大鼠，解剖后取肺组织，把各组织分成3等份，用生理盐水冲洗掉血水，除去水分，装到冻存管内，马上放入-80℃冰箱保存。将收集的接受过低剂量电离射线辐照的WISTAR大鼠肺部组织进行蛋白提取、总蛋白浓度测定、蛋白质变性、凝胶电泳、转膜、免疫反应、化学发光反应、凝胶成像扫描分析、最后按照公式（1）进行α-1抗蛋白酶的相对表达量上调率分析。WISTAR adult male rats weighing between 180 and 220 g were selected and randomly divided into 4 groups, with 6 rats in each group. The control group was instilled with saline and a suspension of silica (the particle size of silica was 200 In the experimental group, uranium ore dust suspension with a concentration of 1.25 mg/ml (the particle size of uranium ore dust is 200 mesh) was instilled through the trachea, 0.2 ml each time, 3 times a week, for 5 consecutive weeks. The rats were killed by cervical dislocation. After dissection, lung tissue was taken, and each tissue was divided into 3 equal parts. The blood was rinsed with normal saline to remove the water, and it was placed in a cryopreservation tube and immediately stored in a -80 ℃ refrigerator. The collected lung tissues of WISTAR rats irradiated with low-dose ionizing rays were subjected to protein extraction, total protein concentration determination, protein denaturation, gel electrophoresis, membrane transfer, immunoreaction, chemiluminescence reaction, gel imaging scanning analysis, Finally, according to formula (1), the relative expression up-regulation rate of α-1 antiprotease was analyzed.

WISTAR成年雄性大鼠气管滴注浓度为1.25 mg/ml的铀矿粉尘悬液5周后，肺部组织中α-1抗蛋白酶的相对表达量上调率为85.6%，此时铀矿粉尘内照射造成的生物损伤等级为Ⅰ级，职业工作人员应增强辐照防护。After the tracheal instillation of 1.25 mg/ml uranium ore dust suspension in WISTAR adult male rats for 5 weeks, the relative expression of α-1 antiprotease in lung tissue was up-regulated by 85.6%. At this time, uranium ore dust was internally irradiated. The level of biological damage caused is Class I, and professional staff should strengthen radiation protection.

实施例2Example 2

选择体重在180 - 220 g之间的WISTAR成年雄性大鼠，随机分4组，每组6只老鼠，对照组气管滴注生理盐水和二氧化硅的悬液（二氧化硅的粒径为200目），实验组分别气管滴注浓度为2.5 mg/ml的铀矿粉尘悬液（铀矿粉尘的粒径为200目），每次滴注0.2 ml，一周滴注3次，连续5周。用颈椎脱臼法处死大鼠，解剖后取肺组织，把各组织分成3等份，用生理盐水冲洗掉血水，除去水分，装到冻存管内，马上放入-80℃冰箱保存。将收集的接受过低剂量电离射线辐照的WISTAR大鼠肺部组织进行蛋白提取、总蛋白浓度测定、蛋白质变性、凝胶电泳、转膜、免疫反应、化学发光反应、凝胶成像扫描分析、最后按照公式（1）进行α-1抗蛋白酶的相对表达量上调率分析。WISTAR adult male rats weighing between 180 and 220 g were selected and randomly divided into 4 groups, with 6 rats in each group. The control group was instilled with saline and a suspension of silica (the particle size of silica was 200 2.5 mg/ml of uranium ore dust suspension (the particle size of uranium ore dust is 200 mesh) in the experimental group, 0.2 ml each time, 3 times a week for 5 consecutive weeks. The rats were killed by cervical dislocation. After dissection, lung tissue was taken, and each tissue was divided into 3 equal parts. The blood was rinsed with normal saline to remove the water, and it was placed in a cryopreservation tube and immediately stored in a -80 ℃ refrigerator. The collected lung tissues of WISTAR rats irradiated with low-dose ionizing rays were subjected to protein extraction, total protein concentration determination, protein denaturation, gel electrophoresis, membrane transfer, immunoreaction, chemiluminescence reaction, gel imaging scanning analysis, Finally, according to formula (1), the relative expression up-regulation rate of α-1 antiprotease was analyzed.

WISTAR成年雄性大鼠气管滴注浓度为2.5 mg/ml的铀矿粉尘悬液5周后，肺部组织中α-1抗蛋白酶相对表达量上调率为158.5 %，此时铀矿粉尘内照射造成的生物损伤等级为Ⅱ级，职业工作人员应及时休息一段时间。After 5 weeks of tracheal instillation of 2.5 mg/ml uranium ore dust suspension in WISTAR adult male rats, the relative expression of α-1 antiprotease in lung tissue was up-regulated by 158.5 %. The level of biological damage is Class II, and professional staff should take a period of rest in time.

实施例3Example 3

选择体重在180 - 220 g之间的WISTAR成年雄性大鼠，随机分4组，每组6只老鼠，对照组气管滴注生理盐水和二氧化硅的悬液（二氧化硅的粒径为200目），实验组分别气管滴注浓度为5.0 mg/ml的铀矿粉尘悬液（铀矿粉尘的粒径为200目），每次滴注0.2 ml，一周滴注3次，连续5周。用颈椎脱臼法处死大鼠，解剖后取肺组织，把各组织分成3等份，用生理盐水冲洗掉血水，除去水分，装到冻存管内，马上放入-80℃冰箱保存。将收集的接受过低剂量电离射线辐照的WISTAR大鼠肺部组织进行蛋白提取、总蛋白浓度测定、蛋白质变性、凝胶电泳、转膜、免疫反应、化学发光反应、凝胶成像扫描分析、最后按照公式（1）进行α-1抗蛋白酶的相对表达量上调率分析。WISTAR adult male rats weighing between 180 and 220 g were selected and randomly divided into 4 groups, with 6 rats in each group. The control group was instilled with saline and a suspension of silica (the particle size of silica was 200 In the experimental group, the uranium ore dust suspension with a concentration of 5.0 mg/ml (the particle size of uranium ore dust is 200 mesh) was instilled through the trachea, 0.2 ml each time, 3 times a week, for 5 consecutive weeks. The rats were killed by cervical dislocation. After dissection, lung tissue was taken, and each tissue was divided into 3 equal parts. The blood was rinsed with normal saline to remove the water, and it was placed in a cryopreservation tube and immediately stored in a -80 ℃ refrigerator. The collected lung tissues of WISTAR rats irradiated with low-dose ionizing rays were subjected to protein extraction, total protein concentration determination, protein denaturation, gel electrophoresis, membrane transfer, immunoreaction, chemiluminescence reaction, gel imaging scanning analysis, Finally, according to formula (1), the relative expression up-regulation rate of α-1 antiprotease was analyzed.

WISTAR成年雄性大鼠气管滴注浓度为5.0 mg/ml的铀矿粉尘悬液5周后，肺部组织中α-1抗蛋白酶相对表达量上调率为245.6 %，此时铀矿粉尘内照射造成的生物损伤等级为Ⅲ级，职业工作人员应调离原工作岗位，并接受适当治疗。After 5 weeks of instillation of 5.0 mg/ml uranium dust suspension in the trachea of WISTAR adult male rats, the relative expression of α-1 antiprotease in lung tissue was up-regulated by 245.6 %. The level of biological damage is grade III, and professional staff should be transferred from their original jobs and receive appropriate treatment.

以上仅仅是本发明的较佳实施方式，根据本发明的上述构思，本领域的熟练人员还可对此作出各种修改和变换。例如，变换铀矿悬液浓度、染毒方式、变换大鼠模型的类型、变换辐照损伤评价体系等等。然而，这些类似的变换和修改均属于本发明的实质。The above are only the preferred embodiments of the present invention, and those skilled in the art can also make various modifications and changes according to the above concept of the present invention. For example, changing the concentration of uranium ore suspension, the way of exposure, changing the type of rat model, changing the radiation damage evaluation system and so on. However, these similar transformations and modifications belong to the essence of the present invention.

Claims (10)

Translated fromChinese

1.一种利用α-1抗蛋白酶A1AT评价铀矿粉尘内照射生物损伤的方法，根据WISTAR大鼠接受气管滴注的铀矿粉尘混悬液的量与肺组织中α-1抗蛋白酶A1AT相对表达量的上调率的剂量-效应关系，通过WISTAR大鼠肺部组织中α-1抗蛋白酶A1AT的相对表达量的上调率，评估铀矿粉尘内照射造成的生物损伤，其特征在于，具体步骤是：1. A method for evaluating the biological damage of uranium ore dust by internal irradiation of uranium ore dust by using α-1 anti-protease A1AT, according to the relative amount of uranium ore dust suspension received by tracheal instillation in WISTAR rats and α-1 anti-protease A1AT in lung tissue The dose-response relationship of the up-regulation rate of the expression level, through the up-regulation rate of the relative expression level of α-1 anti-protease A1AT in the lung tissue of WISTAR rats, to evaluate the biological damage caused by internal irradiation of uranium ore dust, characterized in that the specific steps Yes:（1）建立铀矿混悬液气管滴注WISTAR大鼠模型；(1) Establish a rat model of uranium ore suspension tracheal instillation of WISTAR;（2）采集肺组织；(2) Collect lung tissue;（3）α-1抗蛋白酶A1AT相对表达量上调率的检测；(3) Detection of the relative up-regulation rate of α-1 anti-protease A1AT expression;（4）内照射生物损伤综合评价；(4) Comprehensive evaluation of biological damage from internal irradiation;所述的建立铀矿混悬液气管滴注WISTAR大鼠模型的具体方法是：The specific method for establishing the WISTAR rat model of uranium ore suspension tracheal instillation is:选择体重在180 - 220 g之间的WISTAR成年雄性大鼠，分4组，每组6只老鼠，对照组气管滴注生理盐水和二氧化硅的悬液，实验组分别气管滴注浓度为1.25、2.5和5.0 mg/ml的铀矿粉尘悬液，每次滴注0.2 ml，一周滴注3次，连续5周；WISTAR adult male rats weighing between 180-220 g were selected and divided into 4 groups with 6 rats in each group. The control group was instilled with normal saline and silica suspension, and the experimental group was instilled with a concentration of 1.25. , 2.5 and 5.0 mg/ml of uranium ore dust suspension, instill 0.2 ml each time, 3 times a week for 5 consecutive weeks;所述的采集肺组织的具体方法是：The specific method for collecting lung tissue is:解剖处死的大鼠，并取肺组织，把各组织分成3等份，用生理盐水冲洗掉血水，除去水分，装到冻存管内，马上放入-80℃冰箱保存；The sacrificed rats were dissected, and the lung tissue was taken, and each tissue was divided into 3 equal parts, and the blood was washed with normal saline to remove the water, put into a cryopreservation tube, and immediately stored in a -80°C refrigerator;所述的α-1抗蛋白酶A1AT相对表达量上调率的检测具体方法是：The specific method for detecting the relative expression level of α-1 antiprotease A1AT is as follows:将收集的接受过铀矿粉尘内照射WISTAR大鼠肺部组织进行蛋白提取、总蛋白浓度测定、蛋白质变性、凝胶电泳、转膜、免疫反应、化学发光反应、凝胶成像扫描分析、最后进行α-1抗蛋白酶A1AT的相对表达量上调率分析；The collected lung tissues of WISTAR rats that had been irradiated with uranium ore dust were subjected to protein extraction, total protein concentration determination, protein denaturation, gel electrophoresis, membrane transfer, immunoreaction, chemiluminescence reaction, gel imaging scanning analysis, and finally Analysis of the relative expression up-regulation rate of α-1 antiprotease A1AT;所述内照射生物损伤综合评价的具体方法是：The specific method for the comprehensive evaluation of the internal irradiation biological damage is:当WISTAR大鼠肺部组织中的α-1抗蛋白酶A1AT的相对表达量上调率在30%以内时，表明低剂量伽马射线辐照对生物无损伤效应；当WISTAR大鼠肺部组织中的α-1抗蛋白酶A1AT的相对表达量上调率大于30%而小于100%时，此时的铀矿粉尘内照射造成的生物损伤等级为Ⅰ级；当WISTAR大鼠肺部组织中的α-1抗蛋白酶A1AT的相对表达量上调率大于100%而小于200%时，此时的铀矿粉尘内照射造成的生物损伤等级为Ⅱ级；当WISTAR大鼠肺部组织中的α-1抗蛋白酶A1AT的相对表达量上调率大于200%时，此时的铀矿粉尘内照射造成的生物损伤等级为Ⅲ级。When the relative expression of α-1 antiprotease A1AT in the lung tissue of WISTAR rats was up-regulated within 30%, it indicated that low-dose gamma ray irradiation had no biological damage effect; When the relative expression of α-1 antiprotease A1AT was up-regulated more than 30% but less than 100%, the biological damage level caused by internal irradiation of uranium ore dust was grade I; when α-1 in the lung tissue of WISTAR rats When the relative up-regulation rate of anti-protease A1AT was greater than 100% and less than 200%, the biological damage level caused by internal irradiation of uranium ore dust was grade II; When the relative expression up-regulation rate of uranium ore dust was greater than 200%, the biological damage level caused by internal irradiation of uranium ore dust at this time was grade III.2.根据权利要求1所述的一种利用α-1抗蛋白酶A1AT评价铀矿粉尘内照射生物损伤的方法，其特征在于，2. a kind of method utilizing α-1 anti-protease A1AT to evaluate uranium ore dust internal irradiation biological damage according to claim 1, is characterized in that,所述蛋白提取具体步骤如下：The specific steps of the protein extraction are as follows:在研钵中倒入液氮预冷，迅速取出肺部组织样品，把组织切至均匀置于研钵中，马上加入液氮，进行研磨，在研钵中慢慢加入800 μl LB裂解液，裂解30 min，振荡1-2次，充分裂解，裂解结束后，收集1.5 mL的悬液到EP管中，然后在4℃下，转速12000 r/min，离心20 min后，收集上清液，用树脂沉淀上清液中的蛋白，4℃，12000 r/min离心20 min，去掉上清，晾干，-80℃保存备用，在干燥后的蛋白质团块中加入200-250 μL L3裂解液，用枪头反复吹打直至蛋白质充分分散，超声助溶，4℃，12000 r/min，离心20 min，吸出上清液，转移至新的EP管中。Pour liquid nitrogen into the mortar to pre-cool, quickly take out the lung tissue samples, cut the tissue into the mortar, add liquid nitrogen immediately, grind, and slowly add 800 μl LB lysis solution to the mortar, Lyse for 30 min, shake 1-2 times, and fully lyse. After the lysis, collect 1.5 mL of the suspension into an EP tube, and then centrifuge at 4°C at 12,000 r/min for 20 min, and collect the supernatant. Precipitate the protein in the supernatant with resin, centrifuge at 12,000 r/min for 20 min at 4°C, remove the supernatant, air dry, and store at -80°C for later use. Add 200-250 μL of L3 lysis solution to the dried protein pellet. , Repeatedly pipetting with a pipette tip until the protein was fully dispersed, assisted by ultrasound, centrifuged at 4°C, 12000 r/min for 20 min, aspirated the supernatant, and transferred to a new EP tube.3.根据权利要求1所述的一种利用α-1抗蛋白酶A1AT评价铀矿粉尘内照射生物损伤的方法，其特征在于，3. a kind of method utilizing α-1 anti-protease A1AT to evaluate uranium ore dust internal irradiation biological damage according to claim 1, is characterized in that,所述总蛋白浓度测定的具体步骤如下：The specific steps for the determination of the total protein concentration are as follows:将0.2 mg/ml 的BSA 标准品溶液加稀释液，依次稀释成0、5、10、20、50、100、150和200μg/ml 的标准品溶液，然后测定它们在562 nm下的吸光度值，绘制标准曲线，测定每个样品溶液在562 nm下的吸光度值后，再通过标准曲线计算出样品中蛋白质的浓度。Add the 0.2 mg/ml BSA standard solution to the diluent, dilute it into 0, 5, 10, 20, 50, 100, 150 and 200 μg/ml standard solution in turn, and then measure their absorbance values at 562 nm, Draw a standard curve, measure the absorbance value of each sample solution at 562 nm, and then calculate the protein concentration in the sample through the standard curve.4.根据权利要求1所述的一种利用α-1抗蛋白酶A1AT评价铀矿粉尘内照射生物损伤的方法，其特征在于，4. a kind of method utilizing α-1 anti-protease A1AT to evaluate uranium ore dust internal irradiation biological damage according to claim 1, is characterized in that,所述蛋白质变性的具体步骤如下：The specific steps of the protein denaturation are as follows:将蛋白质溶液按照4:1的比例加入5×蛋白质上样缓冲液，沸水浴变性15 min，放入-20℃冰箱保存备用。The protein solution was added to 5× protein loading buffer at a ratio of 4:1, denatured in a boiling water bath for 15 min, and stored in a -20°C refrigerator for later use.5.根据权利要求1所述的一种利用α-1抗蛋白酶A1AT评价铀矿粉尘内照射生物损伤的方法，其特征在于，5. a kind of method utilizing α-1 anti-protease A1AT to evaluate uranium ore dust internal irradiation biological damage according to claim 1, is characterized in that,所述凝胶电泳的具体步骤如下：The specific steps of the gel electrophoresis are as follows:将30％丙烯酰胺、1.5 M TRIS－HCl、10％SDS、10% 过硫酸铵和TEMED按照一定比例配制成10%的分离胶和5%的浓缩胶；待分离胶凝固后，在电泳槽中加入电泳液，将变性后的蛋白质样品加入电泳孔中，进行电泳，将浓缩胶电压调至75 V，分离胶电压调至100 V，电泳至溴酚蓝刚跑至分离胶底部，即终止电泳，进行转膜。30% acrylamide, 1.5 M TRIS-HCl, 10% SDS, 10% ammonium persulfate and TEMED were prepared into 10% separating gel and 5% stacking gel according to a certain proportion; Add electrophoresis solution, add the denatured protein samples into the electrophoresis wells, conduct electrophoresis, adjust the voltage of the stacking gel to 75 V and the voltage of the separating gel to 100 V, and electrophoresis until the bromophenol blue runs to the bottom of the separating gel, that is, the electrophoresis is terminated. , to transfer the membrane.6.根据权利要求1所述的一种利用α-1抗蛋白酶A1AT评价铀矿粉尘内照射生物损伤的方法，其特征在于，6. a kind of method utilizing α-1 anti-protease A1AT to evaluate uranium ore dust internal irradiation biological damage according to claim 1, is characterized in that,所述转膜的具体步骤如下：The specific steps of the film transfer are as follows:先采用甲醇将PVDF膜进行活化，再在膜的底部放置5-6张7×9 cm的滤纸，再一同放置于转膜仪中，加入转膜液，300 mA恒流转膜30 min，转膜过程中，将转膜槽放置于冰水中降温。First, the PVDF membrane was activated with methanol, and then 5-6 pieces of 7×9 cm filter paper were placed at the bottom of the membrane, and then placed in the membrane transfer apparatus. During the process, place the film transfer tank in ice water to cool down.7.根据权利要求1所述的一种利用α-1抗蛋白酶A1AT评价铀矿粉尘内照射生物损伤的方法，其特征在于，7. a kind of method utilizing alpha-1 anti-protease A1AT to evaluate uranium ore dust internal irradiation biological damage according to claim 1, is characterized in that,所述免疫反应的具体步骤如下：The specific steps of the immune response are as follows:将转好的膜用5%的脱脂牛奶封闭，在脱色摇床上混匀1 h，采用TBST溶解的5%脱脂牛奶对α-1抗蛋白酶一抗Alah3al进行1:1000浓度稀释，然后将其与封闭好的膜进行混匀，4 ℃下孵育过夜，用2% TBST对α-1抗蛋白酶一抗Alah3al孵育过夜的膜进行清洗，室温下在脱色摇床上清洗3次，每次5 min，最后用TBST对二抗羊抗兔稀释8000倍，室温下孵育1.5 h后，在脱色摇床上清洗三次，每次10 min。The transferred membrane was blocked with 5% skim milk, mixed for 1 h on a decolorizing shaker, and the α-1 anti-protease primary antibody Alah3al was diluted 1:1000 with 5% skim milk dissolved in TBST, and then mixed with The blocked membranes were mixed and incubated overnight at 4 °C. The membranes incubated overnight with α-1 anti-protease primary antibody Alah3al were washed with 2% TBST, and washed 3 times on a destaining shaker at room temperature for 5 min each time. The secondary antibody, goat anti-rabbit, was diluted 8000 times with TBST, incubated at room temperature for 1.5 h, and washed three times on a destaining shaker for 10 min each time.8.根据权利要求1所述的一种利用α-1抗蛋白酶A1AT评价铀矿粉尘内照射生物损伤的方法，其特征在于，8. a kind of method utilizing alpha-1 anti-protease A1AT to evaluate uranium ore dust internal irradiation biological damage according to claim 1, is characterized in that,所述化学发光反应的具体步骤如下：The specific steps of the chemiluminescence reaction are as follows:在暗室中将ECLA和ECLB两种试剂在离心管中等体积混合，将PVDF膜的蛋白面朝上，加入混合好的ECL溶液充分反应1-2 min后，放入凝胶成像系统进行扫描分析。Mix the two reagents, ECLA and ECLB, in a centrifuge tube in an equal volume in a dark room, put the protein side of the PVDF membrane up, add the mixed ECL solution and react for 1-2 minutes, then put it into the gel imaging system for scanning and analysis.9.根据权利要求1所述的一种利用α-1抗蛋白酶A1AT评价铀矿粉尘内照射生物损伤的方法，其特征在于，9. a kind of method utilizing alpha-1 anti-protease A1AT to evaluate uranium ore dust internal irradiation biological damage according to claim 1, is characterized in that,所述的凝胶成像扫描分析的具体步骤如下：The specific steps of the gel imaging scanning analysis are as follows:采用凝胶成像系统对蛋白条带进行拍照，再采用Image J软件分析目标带的灰度值，通过对α-1抗蛋白酶A1AT蛋白灰度值扫描获得α-1抗蛋白酶A1AT蛋白表达量，对内参蛋白GAPDH蛋白灰度值扫描获得GAPDH蛋白表达量；将α-1抗蛋白酶A1AT蛋白表达量除以内参蛋白GAPDH蛋白表达量即获得α-1抗蛋白酶A1AT的相对表达量。The protein band was photographed by a gel imaging system, and then the gray value of the target band was analyzed by Image J software. The GAPDH protein expression was obtained by gray value scanning of the internal reference protein GAPDH protein; the relative expression of α-1 antiprotease A1AT was obtained by dividing the expression of α-1 antiprotease A1AT protein by the GAPDH protein expression of the internal reference protein.10.根据权利要求1所述的一种利用α-1抗蛋白酶A1AT评价铀矿粉尘内照射生物损伤的方法，其特征在于，10. a kind of method utilizing alpha-1 anti-protease A1AT to evaluate uranium ore dust internal irradiation biological damage according to claim 1, is characterized in that,所述α-1抗蛋白酶A1AT的相对表达量上调率分析的具体步骤如下：The specific steps for the analysis of the relative expression up-regulation rate of the α-1 antiprotease A1AT are as follows:α-1抗蛋白酶A1AT相对表达量上调率按公式（1）进行计算：The relative up-regulation rate of α-1 antiprotease A1AT was calculated according to formula (1):

(1)。

(1).