【作者】 张晶；

【导师】 裴秋玲；

【作者基本信息】 山西医科大学 ， 卫生毒理学， 2011， 硕士

【摘要】 目的：1.分析比较低浓度砷染毒大鼠红细胞和红细胞膜蛋白的毒性改变，寻找慢性砷中毒早期生物标志物；2.研究低浓度砷染毒大鼠红细胞膜蛋白损伤分子机制，为砷的毒作用机制研究开辟新思路；3.建立低浓度饮用水砷暴露动物模型，测定其毒效应，为国家饮用水砷卫生标准的安全性评价提供依据。方法：140g-160g健康雄性SD大鼠40只，随机分为4组，分别为对照组，10μg/L、60μg/L、360μg/L砷染毒组；3个染毒组采用自由饮用含砷水染毒，对照组饮用普通洁净自来水；染毒30天后，麻醉动物，腹主动脉采集血样，解剖取其主要脏器。分析比较不同组大鼠体重、脏器系数等一般毒性指标。检测不同组大鼠红细胞参数、红细胞衰亡率、红细胞形态及红细胞聚集能力和变形能力等红细胞毒性损伤指标。采用SDS-PAGE电泳和细胞免疫荧光技术分析比较其红细胞膜蛋白变化。结果：1.大鼠一般毒性不同组大鼠同一时间体重差别无统计学意义（P>0.05）；与对照组相比，360μg/L砷染毒组大鼠脾脏脏器系数明显增加，差别有统计学意义（P<0.05）；其余脑、心、肺、肝、肾脏器系数各组之间尚无统计学差异（P>0.05）。2.大鼠红细胞毒性2.1红细胞参数：与正常对照组相比，各染毒组大鼠红细胞参数尚未发生异常改变（P>0.05）。2.2红细胞衰亡率：流式细胞仪检测结果表明，与对照组相比，360μg/L砷染毒组大鼠红细胞衰亡率增加（P<0.05）。2.3红细胞形态：砷染毒组大鼠红细胞形态发生异常改变，细胞表面出现微小突起或明显棘突等不规则变化，360μg/L砷染毒组的变化最为明显，可见多个不规则形红细胞以及明显棘突状红细胞。2.4红细胞血液流变学：与对照组相比，360μg/L砷染毒组的全血低切表观粘度、相对粘度和还原粘度均明显降低（P<0.05）；360μg/L砷染毒组全血中切表观粘度的降低有统计学意义（P<0.05）；同时，360μg/L砷染毒组的红细胞聚集指数也明显小于对照组（P<0.01）；而不同组全血高切表观粘度、相对粘度和还原粘度无统计学改变（P>0.05），红细胞刚性指数和变形指数也无明显变化（P>0.05）。3.大鼠红细胞膜蛋白损伤SDS-PAGE电泳分离得到α-血影蛋白、β-血影蛋白、锚蛋白、带3蛋白、带4.2蛋白、肌动蛋白，共6种主要大鼠红细胞蛋白。与对照组相比，360μg/L砷染毒组大鼠锚蛋白含量减少，差别有统计学意义（P<0.01）。随染毒剂量增加，各组大鼠带3蛋白表达呈现增加趋势，且360μg/L砷染毒组的升高有统计学意义（P<0.05）。其余4种蛋白表达水平的改变均无统计学差异（P>0.05）。免疫荧光检测结果显示，带3蛋白和CD35在砷暴露大鼠红细胞膜上呈现簇集性增加，以60μg/L和360μg/L砷染毒组较为明显。结论：1.低浓度砷染毒可对大鼠红细胞产生毒作用，导致红细胞衰亡率（eryptosis）增加、红细胞形态发生异常改变、红细胞聚集性降低，其原因可能与砷致红细胞膜带3蛋白和锚蛋白含量异常及红细胞膜上带3蛋白和CD35簇集性增加有关。2.低浓度砷暴露大鼠红细胞膜蛋白的损伤较其细胞数量和体积的改变出现更早，红细胞膜蛋白可能是慢性砷中毒的早期生物标志。而脾脏可能是低浓度砷暴露大鼠的早期毒效应靶器官。更多还原

【Abstract】 Objective1. To analyze and compare the changes of the erythrocyte and RBC membrane proteins in ratsexposed low levels arsenic and to explore the potential biomarkers of chronic arsenicpoisoning.2. To study the molecular mechanism of injury on rats erythrocyte membrane proteins in lowlevels arsenic exposure and to open up new avenues for the mechanism of arsenic toxicity.3. To establish the animal model exposed the low levels arsenic in drinking water, to determinethe general toxicity and the RBC membrane damage indices and to provide a basis for thesafety evaluation of national standard for arsenic in drinking water.Methods40 male Sprague- Dawley rats (140g-160g) were divided randomly into 4 groups: controlgroup, 10μg/L As treatment group, 60μg/L As treatment group and 360μg/L As treatment group.The As treatment groups were exposed to arsenic dissolved in drinking water. And the rats ofcontrol group drink ordinary clean tap water. After 30 days of exposure, all animals wereanesthetized and collected blood samples from the abdominal aorta. The major organs wereremoved and weighted. Analysised and compared rats general toxicity indicators including bodyweight, organ weight coefficient in each group. The RBC toxic damage indices were detected indifferent groups, specifically including: red blood cell parameters, eryptosis rate, erythrocytemorphology, erythrocyte deformability and aggregation. The erythrocyte membrane proteinswere seprated and compared by sodium dedecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Immunofluorescence Assay (IFA).Results1. Rats general toxicity indicatorsAt the same time, the weights of rats in different groups have no significant differences(P>0.05). Compared with the control group, in 360μg/L arsenic exposure group spleencoefficients increased significantly, the difference was statistically significant (P<0.05); the restof the brain, heart, lung, liver and kidney coefficients have no significant difference among thegroups (P>0.05). 2. The RBC toxic damage indices2.1 Red Blood Cell ParametersCompared with the control group, the abnormal changes have not occurred in red blood cellparameters of the exposed groups (P>0.05).2.2 Eryptosis RateFlow cytometry results showed that, compared with the control group, eryptosis rateincreased significantly in 360μg/L arsenic exposure group (P<0.05).2.3 Erythrocyte MorphologyThe red blood cell morphology was examined by scanning electron microscopy (SEM). Inthe As treatment groups abnormal shape red blood cells were observed. And the changes in360μg/L As treatment group were particularly obvious, including more irregular-shaped redblood cells and spheroechinocytes.2.4 Erythrocyte Deformability and AggregationErythrocyte rheology test results showed that, compared with the control group, in 360μg/Larsenic exposure group, the low shear whole blood apparent viscosity, relative viscosity andreduced viscosity were significantly decreased (P<0.05). In the 360μg/L As exposure group, theintermediate shear whole blood apparent viscosity was significantly lower (P<0.05). And theerythrocyte aggregation index in 360μg/L As exposure group was significantly smaller than theindex in the control group (P<0.01). The high shear whole blood apparent viscosity, relativeviscosity and reduced viscosity have no significant change in the different groups (P>0.05). Theerythrocyte rigidity index and deformation index also have no significant changes (P>0.05).3. The damage of erythrocyte membrane proteinsSix different proteins, including spectrin-α, spectrin-β, ankyrin, band 3 protein, band 4.2protein and actin, were isolated from the erythrocyte membrane of rats by SDS-PAGE.Compared with the control group, ankyrin content has decreased in the 360μg/L dose group, andthe difference had statistical signification (P<0.01). With exposure levels rising, band 3 proteinexpression presented an increase trend in the treatment groups. And the result showed that theincrease of band 3 was statistically significant in the 360μg/L dose group (P<0.05). Theexpression levels of the remaining four proteins were not significantly changed (P>0.05).Immunofluorescence assay revealed that band 3 protein and CD35 increased and aggregated tothe RBC membrane in rats exposed to arsenic.Conclusions1. Low levels of arsenic exposure can produce toxic effects on rat erythrocyte, resulting inincreased eryptosis rate, abnormal RBC morphology and decreased RBC aggregation. These changes may be related to erythrocyte membrane proteins abnormalities caused by arsenicexposure, which specifically included band 3 and ankyrin levels abnormalities and band 3and CD35 clustering on the erythrocyte membrane.2. The injury of RBC membrane protein appeared earlier than the abnormal changes of RBCnumber and volume in rat of arsenic exposure. Erythrocyte membrane protein may be theearly biological marker in patients of chronic arsenic poisoning. Compared with other organs,spleen may be the early target organ in rats exposed to low levels arsenic.更多还原