镉引起溪蟹糖和蛋白质等物质代谢异常的高血糖激素和应激蛋白基因调控的分子机制研究

Alteration or failure of metabolism has been considered as the initial symptoms of sublethal poisoning. To explore the metabolic responses of freshwater crab Sinopotamon henanense to cadmium (Cd) exposure, key indexes of carbohydrate, protein and lipid metabolisms would be measured and mechanisms would be explored. Enzymes involved in main pathways of carbohydrate metabolism would be assayed. In situ detection of crustacean hyperglycemic hormone (CHH) in X organ-sinus gland and CHH quatitative in hemolymph of the sample will be carried out with the methods of immunohistochemistry and ELISA. Before these detections, CHH gene cloning, preparations of CHH recombinant protein and anti-serum should be completed. For protein metabolism, amino acids mobilization and detoxification protein synthesis will be studied. Real-time PCR is used to analyze the gene expression of detoxificated proteins, i.e., HSP70, SOD and GST. Then, two-dimensional gel electrophoresis and mass spectrometry are used to isolate and identificate the differentially expressed proteins during Cd stress. For lipid metabolism, elements related to lipid storage and key enzymes related to digestion, transfer and synthesis of lipid will be measured. Pyruvate, amino acids and fatty acid derived from the three metabolisms are translated into acetyl coenzyme A, where the cellular respiration begins. Firstly, oxygen consumption and parameters involved in oxygen combining and transport, i.e., oxyhemocyanin and its ratio in hemolymph will be measured. Scondly, ATP levels in tissues will be detected. Thirdly, proteic and gene expression of key enzymes involved in oxygen transport, the citric acid cycle, mitochondrial respiratory chain will be analyzed by spectrophotometry and RT-PCR. Lastly, mitochondrial structure and membrane potential will be observed with transmission electron microscopy and laser scanning confocal microscope. This work would lead to understand Cd-induced high energy demand in the crab and the contributions of carbohydrate metabolism pathway changes to hypoglycemia, then illuminate the interference of Cd with CHH synthesis and excretion. The results also lead to find differentially expressed proteins during Cd stress and are helpful to clarify the toxic mechanisms of Cd and the defence mechanisms of the crab against the toxicity. Biochemical mechanisms focused on Cd-induced alterations of lipid constitutes, storage and function performance would be illustrated by analyzing the relationship between lipid content and transport. In the end, the hypothesis that impairment of mitochondrial energy production derived from carbohydrate, protein and lipid metabolism and the direct damage of Cd to mitochondrial would be testified by the physiological, biochemical and histopathological alterations. This work would be significant for selecting the sensitive biomarker, and improving the biological-evaluated process, further for the bio-monitoring of Cd pollution.

物质和能量代谢是生物体最基本的特征之一。本项目在7项国家自然科学基金资助的基础上，在前期研究积累的前提下，综合利用透射电镜和激光共聚焦、免疫细胞化学与ELISA、双向电泳与质谱、基因克隆、实时荧光定量PCR等方法，以溪蟹为材料，研究镉对糖代谢五条途径中关键酶活性的影响和高血糖激素基因表达调控糖代谢的细胞分子机制；蛋白质代谢酶活性及氨基酸含量和热休克蛋白HSP70、自由基清除蛋白SOD、GST基因的分子保护机制；脂肪代谢中脂肪消化、转运和合成重要酶活性；ATP能量需求和血蓝蛋白mRNA表达、CCO-1基因转录及线粒体膜电位。通过研究，阐明镉对高血糖激素合成和分泌造成干扰或抑制的机理，对蛋白质合成及氨基酸代谢的主要防御机制，在脂肪组成、储备及其功能发挥的生化机理，线粒体功能和血蓝蛋白对氧气利用的调节机制。筛选敏感性强且便于检测的生物效应分子，完善生物评价方法，为水产品健康养殖提供科学依据。

利用显微和亚显微技术、酶活性检测和实时荧光定量等方法，研究了镉处理对河南华溪蟹（Sinopotamon henanense）主要组织糖、蛋白质和脂肪代谢的影响及作用机制，初步掌握了生物大分子的代谢规律，筛选出可作为污染标示物的敏感分子，为重金属检测提供科学依据。主要结果如下：.（1）急性组蛋白质、肝糖原和肌糖原含量下降，游离氨基酸水平、蛋白酶、血淋巴乳酸脱氢酶及谷丙、谷草转氨酶活性增强。亚慢性组血淋巴葡萄糖含量及谷丙、谷草转氨酶活性降低，游离氨和谷氨酰胺含量升高。.（2）急性组血淋巴氧消耗增加，亚慢性组降低；氧合血蓝蛋白的变化类似氧消耗；亚慢性组鳃叶上皮细胞空泡化，鳃叶边缘通道紧缩，上皮细胞高度肿胀且顶膜微绒毛减少，鳃血腔缩小，线粒体嵴消失，细胞核膜变形，核染色质浓缩且边集化；急性和亚慢性组镉蓄积量和SOD活性无显著差异；急性组金属硫蛋白（MT）含量显著高于亚慢性组；MDA含量变化与MT相反。.（3）急性组G-6-PDH活性、NADPH和GSH含量及GSH/GSSG比值下降；亚慢性组G-6-PDH活性和NADPH含量显著上升，GSH先升后降，GSSG随浓度增加显著上升，GSH/GSSG比值下降。.（4）心脏CCO活性和cco-1 mRNA表达水平下调；急性组ATP含量和IDH活性升高，LDH活性下降；亚慢性组则相反。急性组肌肉组织ATP含量未发生明显变化，亚慢性组则显著下降；IDH、CCO、LDH活性均未发生显著变化，亚慢性组显著下降；cco-1 mRNA表达急性组低浓度显著上调，但高浓度下调至对照组水平；ldh mRNA表达在急性组显著上调。cco-1和ldh mRNA表达在亚慢性最高浓度组皆显著下调。.（5）脂肪含量随镉浓度增加和时间延长逐渐降低，降低卵巢和肝胰腺指数。镉处理10d，血淋巴甘油三酯含量、组织NADPH和ATP含量及胰脂肪酶（PL）活性升高。然而，脂蛋白脂肪酶（LPL）活性降低导致脂肪损伤。15d 和20d，脂肪含量和淋巴液甘油三酯水平进一步降低。NADPH和ATP含量、PL和LPL等活性降低，进一步导致低水平脂肪积累。.本项目的完成共发表论文23篇，其中SCI收录15篇，二区6篇；获得发明专利3项。毕业博士2名，硕士5名，出站博士后1名。2人获研究生国家奖学金，5人获研究生学业奖学金。此外，举办学术报告会10余场。