锯缘青蟹黄水病的血液指标及组织基因表达差异研究
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摘要
本论文对锯缘青蟹(Scylla serrata)“黄水病”进行了研究,该病是近些年来发生在我国浙江沿海养殖锯缘青蟹的一种流行性疾病,患病青蟹的主要症状表现为甲壳内出现黄色腔液、血淋巴呈黄色或浊白不凝液体,该病持续时间较长,呈季节性流行趋势,危害严重。为了更多地了解该病的致病机理,我们对患病蟹的免疫水平、血液生化指标进行了测定,并对肝胰腺在“黄水病”胁迫条件下的基因表达差异进行了探索性研究。本研究主要结果如下:
1.对锯缘青蟹的血细胞进行分类
以正常青蟹血淋巴为研究材料,分别进行显微和亚显微观察,将青蟹血细胞分为三类:大颗粒细胞、小颗粒细胞和无颗粒细胞,三类血细胞在光镜和电镜下具有明显不同的结构特征,这些特征与其在免疫过程中可能承担的功能相关。对血细胞进行分类,为分析“黄水病”的血细胞比例变化提供理论前提。
2.“黄水病”对锯缘青蟹免疫水平的影响
选择血细胞密度(THC)、血细胞比例、血细胞吞噬活性、血清酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、溶菌酶(LZM)、过氧化物酶(POD)、超氧化物歧化酶(SOD)、酚氧化酶(PO)作为指标,对“黄水病”青蟹和健康青蟹的免疫水平进行测定比较,发现大多数免疫指标发生显著变化,其中血细胞密度、血细胞吞噬活性和绝大多数血清酶活性指标皆极显著下降,血细胞比例也发生了显著变化,说明“黄水病”发生后,青蟹的免疫能力受到抑制,免疫系统可能遭到破坏,受到继发感染的可能性增加。
3.“黄水病”锯缘青蟹血液生化分析
与健康青蟹相比,“黄水病”锯缘青蟹血液中大多数生化指标发生了显著改变,病蟹血清蛋白质含量及部分血清酶活性皆极显著降低,血清中糖类、脂类及蛋白质代谢物含量异常,电解质紊乱,说明患病青蟹的消化吸收、排泄、渗透压调节等功能遭到破坏,与这些功能相关的组织发生严重病变。
4.“黄水病”锯缘青蟹与健康青蟹肝胰腺组织基因表达差异
采用分子病理学手段研究了“黄水病”青蟹肝胰腺组织的病理变化。通过mRNA差异显示技术,发现病蟹与健康蟹肝胰腺组织的基因表达存在差异。对不同来源的肝胰腺组织的表达基因片段进行PCR扩增和PAGE比较筛选,发现了至少10条差异条带,其中有2条被成功回收和重扩增:一条来自健康青蟹组织,另一条来自病蟹组织,认为是差异表达的基因片段。对该两条基因片段进行克隆测序,将测序结果在GenBank数据库进行比对,未发现相匹配基因,不排除其来自某些暂未研究到的新基因的可能,这些基因分别在健康青蟹和病蟹肝胰腺中表达,在不同的生理状态下开启或关闭,微观分子水平上的变化,引起宏观机体组织的代谢变化。该研究从分子层面上揭示病理变化情况,这也为病理研究提供新的思路。
This study investigate a kind of epidemic disease——“yellow water”disease which broke out in the cultured Scylla sarrata along the coastal area of Zhejaing province,China in these years.The mian symptoms of the diseased crab display as following:yellow water could be seen under carapace,hemolymph turned into yellow or turbid white liquid which can’t coagulated.The disease caused serious loss because of its long duration,and it shows a seasonal epidemic trend now.In order to learn more about the pathogenesis of the disease,we survey and evaluate the immunization levels and measured blood parameters of diseased crabs,and carry out a exploratory reseach about the mRNA differential displays in hepatopancreas from diseased crabs which coerced by“yellow water”disease.The main results of our study are as followings:
1. Classified the hemocytes of Scylla serrata into three categories
Use the hemolymph of healthy crab as studied material, and observated it microscopically and submicroscopically.We classified the hemocytes of Scylla serrata into three categories: semigranular cell(SGC),granular cell(GC) and hyaline cell(HC),these three kinds of hemocytes show significantly differences microscopic and submacroscopic structures.Classification of the hemocytes can provide the theory premise for the analysis of the changes in the hemocyte proportion.
2.The impact of“yellow water”disease on the immunization levels of scally serrata.
Chosed total hemocyte count(THC),hemocyte proportion,the phagocytosis activity of hemocytes,ACP, AKP, LZM, POD, SOD, PO activity in serum as indicators,measuered and compared the immunization levels of crab with“yellow water”disease and controls,and discoverd that most of the indicators showed significantly changes,the THC,hemocyte and phagocytosis activity and enzyme activity in serum decreased significantly,the proportion of different hemocytes also changed significantly.All these indicated that after contracting“yellow water”disease,the capacity of immunity was inhibited,the immunity system suffered damage,and the possibility of contracting secondary infection would increase.
3.Blood biochemistry analysis of“yellow water”disease in Scylla serrata.
Compared to healthy crabs,most of the blood biochemical parameters of the diseased ones changed significantly.The protein content and enzyme activity in serum decreased significantly,the metabolites contents of carbohydrate/lipid/protein in serum is abnormal,and electrolyte in serum imbalanced.These indicated that,the capacity of digestion/absorption/excretion and Osmoregulation had been damaged,and the organs associated with these function had serious lesion.
4.mRNA differential displays in hepatopancreas of crabs with“yellow water”disease and controls.
Adopt the methold of molecular pathology and study the pathological changes in the hepatopancreas of crabs with“yellow water”disease.Using the mRNA differential display technology,we discovered the exist of differential displayed genes in diseased crabs and healthy ones.Amplificated the genes of hepatopancreas from different crabs using PCR technology,and compared the PCR product using PAGE. At least 10 differential displayed strips were discovered,and two of them wre recycled and reamplificated successfully:one from healthy crabs and another from diseased crabs,which were thought to be the fragments of differential diaplayed genes.Cloned and sequenced the two gene fragments,blasted the results in GenBank,and no similar gene were found,thought that they may be new genes which were respectively displayed in healthy crabs and diseased crabs,they were opened or closed in different physiological states.The changes happened in micro-molecular level lead the changes of metabolism in macro-tissues,and revealed the pathological changes on molecular level,and this study can also provide new ideas in pathological reseach.
1.对锯缘青蟹的血细胞进行分类
以正常青蟹血淋巴为研究材料,分别进行显微和亚显微观察,将青蟹血细胞分为三类:大颗粒细胞、小颗粒细胞和无颗粒细胞,三类血细胞在光镜和电镜下具有明显不同的结构特征,这些特征与其在免疫过程中可能承担的功能相关。对血细胞进行分类,为分析“黄水病”的血细胞比例变化提供理论前提。
2.“黄水病”对锯缘青蟹免疫水平的影响
选择血细胞密度(THC)、血细胞比例、血细胞吞噬活性、血清酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、溶菌酶(LZM)、过氧化物酶(POD)、超氧化物歧化酶(SOD)、酚氧化酶(PO)作为指标,对“黄水病”青蟹和健康青蟹的免疫水平进行测定比较,发现大多数免疫指标发生显著变化,其中血细胞密度、血细胞吞噬活性和绝大多数血清酶活性指标皆极显著下降,血细胞比例也发生了显著变化,说明“黄水病”发生后,青蟹的免疫能力受到抑制,免疫系统可能遭到破坏,受到继发感染的可能性增加。
3.“黄水病”锯缘青蟹血液生化分析
与健康青蟹相比,“黄水病”锯缘青蟹血液中大多数生化指标发生了显著改变,病蟹血清蛋白质含量及部分血清酶活性皆极显著降低,血清中糖类、脂类及蛋白质代谢物含量异常,电解质紊乱,说明患病青蟹的消化吸收、排泄、渗透压调节等功能遭到破坏,与这些功能相关的组织发生严重病变。
4.“黄水病”锯缘青蟹与健康青蟹肝胰腺组织基因表达差异
采用分子病理学手段研究了“黄水病”青蟹肝胰腺组织的病理变化。通过mRNA差异显示技术,发现病蟹与健康蟹肝胰腺组织的基因表达存在差异。对不同来源的肝胰腺组织的表达基因片段进行PCR扩增和PAGE比较筛选,发现了至少10条差异条带,其中有2条被成功回收和重扩增:一条来自健康青蟹组织,另一条来自病蟹组织,认为是差异表达的基因片段。对该两条基因片段进行克隆测序,将测序结果在GenBank数据库进行比对,未发现相匹配基因,不排除其来自某些暂未研究到的新基因的可能,这些基因分别在健康青蟹和病蟹肝胰腺中表达,在不同的生理状态下开启或关闭,微观分子水平上的变化,引起宏观机体组织的代谢变化。该研究从分子层面上揭示病理变化情况,这也为病理研究提供新的思路。
This study investigate a kind of epidemic disease——“yellow water”disease which broke out in the cultured Scylla sarrata along the coastal area of Zhejaing province,China in these years.The mian symptoms of the diseased crab display as following:yellow water could be seen under carapace,hemolymph turned into yellow or turbid white liquid which can’t coagulated.The disease caused serious loss because of its long duration,and it shows a seasonal epidemic trend now.In order to learn more about the pathogenesis of the disease,we survey and evaluate the immunization levels and measured blood parameters of diseased crabs,and carry out a exploratory reseach about the mRNA differential displays in hepatopancreas from diseased crabs which coerced by“yellow water”disease.The main results of our study are as followings:
1. Classified the hemocytes of Scylla serrata into three categories
Use the hemolymph of healthy crab as studied material, and observated it microscopically and submicroscopically.We classified the hemocytes of Scylla serrata into three categories: semigranular cell(SGC),granular cell(GC) and hyaline cell(HC),these three kinds of hemocytes show significantly differences microscopic and submacroscopic structures.Classification of the hemocytes can provide the theory premise for the analysis of the changes in the hemocyte proportion.
2.The impact of“yellow water”disease on the immunization levels of scally serrata.
Chosed total hemocyte count(THC),hemocyte proportion,the phagocytosis activity of hemocytes,ACP, AKP, LZM, POD, SOD, PO activity in serum as indicators,measuered and compared the immunization levels of crab with“yellow water”disease and controls,and discoverd that most of the indicators showed significantly changes,the THC,hemocyte and phagocytosis activity and enzyme activity in serum decreased significantly,the proportion of different hemocytes also changed significantly.All these indicated that after contracting“yellow water”disease,the capacity of immunity was inhibited,the immunity system suffered damage,and the possibility of contracting secondary infection would increase.
3.Blood biochemistry analysis of“yellow water”disease in Scylla serrata.
Compared to healthy crabs,most of the blood biochemical parameters of the diseased ones changed significantly.The protein content and enzyme activity in serum decreased significantly,the metabolites contents of carbohydrate/lipid/protein in serum is abnormal,and electrolyte in serum imbalanced.These indicated that,the capacity of digestion/absorption/excretion and Osmoregulation had been damaged,and the organs associated with these function had serious lesion.
4.mRNA differential displays in hepatopancreas of crabs with“yellow water”disease and controls.
Adopt the methold of molecular pathology and study the pathological changes in the hepatopancreas of crabs with“yellow water”disease.Using the mRNA differential display technology,we discovered the exist of differential displayed genes in diseased crabs and healthy ones.Amplificated the genes of hepatopancreas from different crabs using PCR technology,and compared the PCR product using PAGE. At least 10 differential displayed strips were discovered,and two of them wre recycled and reamplificated successfully:one from healthy crabs and another from diseased crabs,which were thought to be the fragments of differential diaplayed genes.Cloned and sequenced the two gene fragments,blasted the results in GenBank,and no similar gene were found,thought that they may be new genes which were respectively displayed in healthy crabs and diseased crabs,they were opened or closed in different physiological states.The changes happened in micro-molecular level lead the changes of metabolism in macro-tissues,and revealed the pathological changes on molecular level,and this study can also provide new ideas in pathological reseach.
引文
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