镉对河南华溪蟹血淋巴细胞的毒性效应及脂多糖的调节作用

发布时间：2018-08-06 17:52

【摘要】：本博士学位论文以河南华溪蟹(Sinopotamon henanense)的血淋巴细胞(hemocytes)为材料,研究了亚慢性镉染毒对血淋巴细胞的毒性作用及免疫增强剂脂多糖的调节作用。实验设置了3个镉处理组(0.725、1.450和2.900 mg/L)和一个空白对照组,分别处理7 d、14 d和21 d。染毒后将蟹体平均分为两组,其中一组个体用来研究镉的毒性作用,另一组个体分别注射10μg/mL浓度的脂多糖用来研究脂多糖对镉毒性的调节作用。首先,采用透射电镜技术(transmission electron microscope,TEM)观察了血淋巴细胞的形态结构变化及膜的完整性,从形态学角度探究镉对血淋巴细胞超微结构的损伤。其次,用血球计数板统计了血淋巴细胞的总数(total hemocyte counts,THC);采用火焰原子吸收仪检测了血淋巴细胞中镉的富集量;用比色法测定了活性氧自由基(reactive oxygen species,ROS)水平,酸性磷酸酶(acid phosphatase,ACP)、碱性磷酸酶(alkaline phosphatase,AKP)和溶菌酶(lysozyme,LSZ)等溶酶体酶的活性;利用中性红染色法测定了溶酶体膜的稳定性,系统研究了由于镉的富集引起ROS水平升高而进一步介导的血淋巴细胞生理生化功能的变化,以及脂多糖对这种变化的调节作用。在此基础上,为了研究抗氧化酶活性和氧化损伤程度,用购置的生化试剂盒测定了总抗氧化能力(total antioxidant capacity,T-AOC)、抗氧化酶:超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase,GPx)和过氧化物酶(peroxidase,POD)活性的变化及脂质过氧化产物丙二醛(malondialdehyde,MDA)的含量。此外,采用2,4-二硝基苯肼(DNPH)比色法测定了蛋白质羰基(protein carbonyl derivates,PCO)含量,用KCL-SDS沉淀法检测了DNA-蛋白质交联率(DNA-protein crosslink,DPC)。最后,利用实时荧光定量PCR法对免疫相关基因酚氧化酶原(prophenoloxidase,简称proPO)、溶菌酶(lysozyme,简称LSZ)和金属硫蛋白(metallothionein,简称MT)三种基因mRNA表达水平的变化,用比色法测定了PO的活性,从分子水平上研究了镉胁迫的应答机制及脂多糖的调节作用,阐述了镉对河南华溪蟹免疫系统的毒性作用及致毒机制。研究结果包括以下四个方面:1.镉对河南华溪蟹血淋巴细胞超微结构的影响根据细胞的整体形态、胞质中的颗粒数目及其大小可将河南华溪蟹血淋巴细胞分为三类:大颗粒细胞、小颗粒细胞和无颗粒细胞。其中大颗粒细胞的体积较大、细胞核较小、胞质中有较多的电子密度颗粒,而且各类细胞器较多;小颗粒细胞体积稍小,胞质中含少量颗粒;无颗粒细胞体积一般较小,几乎无颗粒,较少观察到细胞器。镉暴露可以引起溪蟹颗粒细胞进行脱颗粒,细胞核变形、染色质浓缩,线粒体出现了嵴断裂、空泡化甚至消失的现象,粗面内质网的腔扩张、其上的核糖体脱落,溶酶体膜模糊甚至破裂。另外还发现,高浓度组(2.900 mg/L)镉暴露时间由7 d延长至21 d时,血淋巴细胞超微结构的损伤程度较低浓度组(0.725 mg/L)的严重。2.镉处理对河南华溪蟹血淋巴细胞免疫相关因子的影响及脂多糖的调节作用河南华溪蟹血淋巴细胞镉的富集量随着染毒浓度的增加和暴露时间的延长,呈现上升趋势,并且有剂量-效应。镉导致溪蟹血淋巴细胞总数显著下降,ROS水平显著上升,溶酶体膜的稳定性下降。除LSZ外,镉暴露引起了血淋巴细胞中的ACP和AKP活性均升高;镉染毒后再注射脂多糖,与镉单独作用相比,脂多糖引起溪蟹血淋巴细胞总数下降,ROS在一定程度上降低,溶酶体膜的稳定性也下降,ACP和AKP活力均升高,LSZ活力无显著变化。3.镉胁迫对河南华溪蟹血淋巴细胞抗氧化酶和氧化损伤的影响及脂多糖的调节作用河南华溪蟹血淋巴细胞中抗氧化酶SOD、CAT、GPx和POD的活性随着镉浓度的增加和处理时间的延长而整体上呈现出“先升高后降低”的趋势,即低浓度镉(0.725 mg/L)显著诱导抗氧化酶活性升高,高浓度镉(2.900 mg/L)抑制了酶活性。脂多糖作用后,四种酶活性在不同程度上均低于其相应的镉处理组的活性。血淋巴细胞中的氧化损伤指标:PCO、DPC和MDA含量均呈现逐渐升高趋势。与镉单独作用相比,镉暴露后再注射脂多糖,三个氧化损伤指标出现不同程度的下降,然而镉暴露时间延长至21 d时,各氧化损伤指标无显著下降趋势,脂多糖在一定程度上减轻镉导致的氧化损伤效应。4.河南华溪蟹血淋巴细胞对镉的免疫应答及脂多糖的调节作用随着镉浓度的增加或者暴露时间的延长,河南华溪蟹血淋巴细胞中proPO mRNA的表达量在整体上呈现下降趋势,与镉单独作用相比,脂多糖作用后,proPO mRNA的表达量升高。PO活性在整个处理过程中呈现“先升后降”趋势。镉暴露后注射脂多糖,与相应的镉单独处理组相比,PO活性在整体上呈现下降趋势。镉诱导溪蟹血淋巴细胞中的LSZ mRNA表达水平均升高,并呈现出剂量-效应。镉暴露后注射脂多糖,与相应的镉单独处理组相比,LSZ mRNA表达水平出现不同程度的下降趋势。此外,随着暴露时间的延长或者镉浓度的增加,MT mRNA的表达水平有升高趋势,并呈现时间和剂量-效应。镉暴露后再注射脂多糖时,与相应的镉作用组相比,MT mRNA的表达水平呈下降趋势。研究结果表明:1.镉胁迫改变了河南华溪蟹血淋巴细胞的超微结构,其中高浓度镉暴露对血淋巴细胞造成的损伤更严重,说明血淋巴细胞是镉毒性作用的靶器官,这些结构上的损伤可能会影响免疫相关因子正常的合成和分泌,导致溪蟹失去正常的免疫功能。2.由于镉在河南华溪蟹血淋巴细胞的富集而引起了ROS水平的升高,而ROS进一步对溶酶体造成损坏,释放溶酶体酶;而脂多糖会降低ROS水平,在一定程度上能增强蟹体的免疫能力。3.镉可以通过改变河南华溪蟹血淋巴细胞抗氧化酶活性,进而诱导脂质、蛋白质和DNA发生氧化损伤,最终使血淋巴细胞免疫功能下降。脂多糖可以调节抗氧化酶活性,使氧化损伤指标降低。4.镉对河南华溪蟹血淋巴细胞免疫相关基因有明显的影响,镉抑制了proPO mRNA的表达,却能诱导LSZ和MT两种基因mRNA的表达,而脂多糖对这三种基因表达的调节作用不同。
[Abstract]:In this doctoral dissertation, the toxic effects of subchronic cadmium exposure on blood lymphocytes and the regulation of lipopolysaccharide were studied with the blood lymphocyte (hemocytes) of Sinopotamon henanense in Henan. The experiment set up 3 cadmium treatment groups (0.725,1.450 and 2.900 mg/L) and a blank control group, respectively, to deal with 7 D, 14 d and 21 D. were divided into two groups, one of which was used to study the toxicity of cadmium. The other group was injected with 10 g/mL concentration of lipopolysaccharide to study the regulation of lipopolysaccharide on cadmium toxicity. First, the blood lymphocytes were observed by transmission electron microscopy (transmission electron microscope, TEM). Morphological changes and membrane integrity were used to investigate the damage of cadmium to the ultrastructure of blood lymphocytes from the morphological angle. Secondly, the total number of blood lymphocytes (total hemocyte counts, THC) was measured by the blood cell count board, and the concentration of cadmium in blood lymphocytes was detected by flame atomic absorptiometer, and the reactive oxygen free radicals were measured by colorimetric method. The activity of lysosomal enzymes (reactive oxygen species, ROS), acid phosphatase (acid phosphatase, ACP), alkaline phosphatase (alkaline phosphatase, AKP), and lysozyme (lysozyme, LSZ)). The stability of the lysosomal membrane was determined by neutral red staining. The system was further mediated by the enrichment of cadmium. Changes in the physiological and biochemical functions of blood lymphocytes and the regulation of lipopolysaccharide on this change. On this basis, in order to study the activity of antioxidant enzymes and the degree of oxidative damage, the total antioxidant capacity (total antioxidant capacity, T-AOC), antioxidant enzyme: superoxide dismutase (superoxide dismuta) were measured by the purchased biochemical kit. Se, SOD), the changes in the activity of catalase (catalase, CAT), glutathione peroxidase (glutathione peroxidase, GPx) and peroxidase (peroxidase, POD), and the content of malondialdehyde (malondialdehyde, MDA) in the product of lipid peroxidation. Vates, PCO) content, the DNA- protein crosslinking rate (DNA-protein crosslink, DPC) was detected by KCL-SDS precipitation method. Finally, three kinds of genes were expressed by the real-time fluorescent quantitative PCR method for the immunologic related genes of phenoloxidase (prophenoloxidase, proPO), and the lysozyme (lysozyme, LSZ) and metallothionein. The activity of PO was measured by colorimetric method. The response mechanism of cadmium stress and the regulation of lipopolysaccharide were studied at the molecular level, and the toxic and toxic mechanisms of cadmium on the immune system of Henan crabs were expounded. The results included the following four aspects: 1. the influence of cadmium on the ultrastructure of the blood lymphocyte of Creek crabs According to the overall morphology of the cell, the number and size of the particles in the cytoplasm can be divided into three types: large granulosa cells, small granular cells and no granulosa cells in Henan. Large granular cells have larger size, smaller nuclei, more electron density particles in the cytoplasm, and many kinds of organelles; small granular cell bodies. The volume of the cytoplasm is small and the cytoplasm contains a small amount of particles; the volume of the granulosa cells is generally small and almost no particles. The cytoplasm is less observed. Cadmium exposure can cause degranulation, nuclear deformation, chromatin concentration, mitochondrial crista fracture, vacuolization to the disappearance, the dilation of the rough endoplasmic reticulum, and ribose on the cytoplasm. It was also found that the cadmium exposure time of the high concentration group (2.900 mg/L) was prolonged from 7 d to 21 d, and the damage degree of the ultrastructure of the blood lymphocyte was more than that of the low concentration group (0.725 mg/L), the effect of.2. treatment on the blood lymphatic cell immunity related factors of Henan Chinese crabs and the regulation of lipopolysaccharide The concentration of cadmium in the blood lymphocytes of crabs of South China brook increased with the increase of exposure concentration and exposure time, which showed an upward trend and had a dose effect. Cadmium led to a significant decrease in the total number of blood lymphocytes in crabs, the ROS level increased significantly, and the stability of the lysosome membrane decreased. In addition to LSZ, cadmium exposure caused ACP and AKP activities in blood lymphocytes. Compared with cadmium alone, lipopolysaccharide caused the decrease of the total number of blood lymphocytes in crabs, ROS decreased to a certain extent, the stability of the lysosome membrane decreased, the activity of ACP and AKP increased, and the activity of LSZ had no significant changes in.3. stress on the antioxidant enzymes and oxidative damage of the blood lymphocytes of Henan Chinese crabs. The effects of injury and the regulation of lipopolysaccharide on the activity of antioxidant enzymes SOD, CAT, GPx and POD in the blood lymphocytes of Henan Chinese crabs with the increase of cadmium concentration and the prolongation of treatment time, the trend of "first increase and then decrease" was presented as a whole, that is, low concentration of cadmium (0.725 mg/L) significantly induced the increase of antioxidant enzyme activity and high concentration of cadmium (2.900 mg/). L) inhibited the activity of the enzyme. After lipopolysaccharide, the activity of the four enzymes was lower than the activity of the corresponding cadmium treatment group. The oxidative damage indexes in the blood lymphocytes, such as PCO, DPC and MDA, were all increasing gradually. Compared with the cadmium alone, the cadmium was reinjected into lipopolysaccharide after exposure to cadmium, and the three oxidative damage indexes appeared in different processes. When the exposure time of cadmium was prolonged to 21 d, there was no significant decrease in the oxidative damage index, and the lipopolysaccharide alleviated the oxidative damage effect caused by cadmium to a certain extent,.4. Henan Chinese crab blood lymphocytes to the immune response to cadmium and the regulation of lipopolysaccharide with the increase of cadmium concentration or prolongation of exposure time, Henan Hua Hua. The expression of proPO mRNA in the blood lymphocytes of crabs showed a downward trend on the whole. Compared with the effect of cadmium alone, the expression of proPO mRNA increased after the action of lipopolysaccharide, and the.PO activity showed a "first rise and then descend" trend during the whole process. After cadmium exposure, the activity of PO was compared with that of the isolated treatment group. The level of LSZ mRNA expression in the blood lymphocytes of crabs induced by cadmium increased and showed a dose effect. After exposure to cadmium exposure to lipopolysaccharide, the expression level of LSZ mRNA decreased in different degrees. In addition, with the prolongation of exposure time or the increase of cadmium concentration, MT mRNA The expression level of MT mRNA was decreased when cadmium exposed to lipopolysaccharide after exposure to cadmium. The results showed that 1. cadmium stress changed the ultrastructure of the blood lymphocytes of Henan Chinese crabs, which caused by high concentration of cadmium exposure to blood lymphocytes. The damage is more serious, indicating that the blood lymphocyte is the target organ of the toxic effect of cadmium, the damage of these structures may affect the normal synthesis and secretion of immune related factors, which leads to the loss of normal immune function of the crabs,.2. increases the level of ROS because of the enrichment of cadmium in the blood lymphocytes of Henan crabs, and the ROS is further to the lysase. The body causes damage and release lysosomal enzyme, while lipopolysaccharide can reduce the ROS level and enhance the immune ability of the crab body to a certain extent,.3. cadmium can induce lipid, protein and DNA oxidative damage by changing the antioxidant enzyme activity of the blood lymphocytes of Henan crabs, and the immune function of blood lymphocytes can be reduced at the end of the year. The effects of.4. cadmium on the immune related genes in the blood lymphocytes of Henan Chinese crabs were obviously affected by the regulation of antioxidant enzyme activity. Cadmium inhibited the expression of proPO mRNA, but could induce the expression of LSZ and MT two genes mRNA, and the regulation of lipopolysaccharide on the expression of the three genes was different.
【学位授予单位】：山西大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R114

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