【作者】 张惠娟；

【导师】 谢从新；

【作者基本信息】 华中农业大学 ， 水产养殖学， 2011， 博士

【摘要】 黑斑原鮡(Glyptosternum maculatum)主要分布于雅鲁藏布江中游干支流,隶属于鲇形目(Siluriformes),鮡科(Sisoridae),原鮡属(Glyptosternum),是鰋鮡鱼类中的一种。该鱼除在腹腔内具有正常肝脏外,在体侧肌肉与皮肤之间还有一特殊组织,位于胸鳍基部后方的皮下与肌肉间,由一带状组织(连接带)与腹腔内肝脏相连接。经研究证实这一组织并非畸形所致,为黑斑原鮡特有组织——“腹腔外肝”。本文主要研究了黑斑原鮡早期肝脏的发生；腹腔内肝和腹腔外肝的生理生化差异；鮡科鱼类的肝脏特征及黑斑原鮡的血液学。论文主要结果如下：1.黑斑原鮡的肝脏首先出现在原始消化管(食道部位)的左侧,肝脏发生的过程分为三个阶段：(1)无腹腔外肝阶段；(2)出现肝脏“突起”阶段；(3)腹腔外肝出现阶段。腹腔外肝是在腹腔壁肌肉层增厚,食道和骨鳔增大的过程中,首先形成较为粗大的突起,然后该突起基部和中部逐渐变细形成连接带,而端部膨大形成腹腔外肝。经研究表明,腹腔外肝与腹腔内肝脏一样位于腹腔,故建议将“腹腔外肝”改为“副肝”(attaching liver),‘腹腔内肝脏”相应的改为“主肝’’(main liver)。2.黑斑原鮡主肝和副肝的结构在显微和超微水平上都基本一致。肝脏连接带的结构也与主肝和副肝的结构相似。组织学中,连接带中的静脉,动脉和胆管均比较粗大,这表明连接带是主肝和副肝进行物质活动的传输纽带。3.对黑斑原鮡肝脏(主肝和副肝)的三种组成成分(蛋白质、糖原、脂肪)的含量、三种代谢酶(LDH、AST、ALT)的活性、两种抗氧化酶(SOD、CAT)及总抗氧化能力进行了测定。主肝中的蛋白质含量、脂肪含量、总抗氧化能力、超氧化物歧化酶活性和过氧化氢酶活性均略高于副肝；而主肝中的糖原含量、乳酸脱氢酶活性、谷草转氨酶活性和谷丙转氨酶活性均略低于副肝,但主肝和副肝在各指标上的差异均未达到显著水平(P>0.05)。4.获得了β-actin、Cu-Zn SOD、Mn SOD和CAT四个基因的特异性片段,并以β-actin为参照基因得到了主肝和副肝中Cu-Zn SOD、Mn SOD和CAT mRNA的相对表达量。黑斑原鮡主肝和副肝中Cu-Zn SOD、Mn SOD和CAT mRNA表达量的差异均呈显著水平(P<0.05),主肝Cu-Zn SOD、Mn SOD和CAT mRNA的相对表达量均显著大于副肝。5.本研究共解剖18种(10属)鮡科鱼类,其中7种(7属)具有明显的副肝,另外的11种(4属)不具有副肝。不同属之间存在肝脏形态的差异。胚属、黑鮡属和纹胸鮡属的种类均不具有副肝,而鰋鮡鱼类均具有副肝。而同属的不同种之间也存在着肝脏形态的差异。褶鮡属的黄斑褶鮡和间褶鮡肝脏形态差别较大,黄斑褶鮡具有副肝,而间褶鮡仅具有明显的肝脏突起。副肝与主肝重量之比按从小到大的顺序依次为：黄斑褶鮡、拟鰋属<鮡属<石爬鮡属<原鮡属<鰋属<凿齿鮡属。6.根据鮡科鱼类的地理分布界域和副肝的相对大小与分布海拔所做的相关性分析结果(极显著正相关),可以看出随着分布海拔的升高,鮡科鱼类肝脏的发展趋势为：无副肝(魾属和黑鮡属)→出现肝脏突起(纹胸鮡属)→肝脏突起非常明显(间褶鮡)→副肝出现(黄斑褶鮡)→副肝逐渐增大(鰋鮡鱼类)。副肝是鮡科鱼类在适应高海拔,急流环境的过程中,随体形的变化形成的,它实际上是对高海拔、急流这一特殊环境的适应。7.根据副肝从无到有的形态特征变化,鮡科鱼类总的演化趋势为(黑鮡属,魾属,纹胸鮡属)→褶鮡属→鰋鮡鱼类。依据副肝从小到大的形态特征分析,鰋鮡鱼类在系统进化上具有以下演化趋势：拟鰋属Pseudexostoma(副肝明显小于主肝)→鮡属Pareuchiloglanis(副肝小于主肝,但较拟鰋属大)→石爬鮡属Euchiloglanis(副肝小于主肝,但较鮡属大)→原鮡属Glyptosternum(副肝小于主肝,但较石爬鮡属大)→鰋属Exostoma(副肝与主肝几乎等大)→凿齿鮡属Glaridoglanis(副肝大于主肝)。8.对黑斑原鮡的红细胞数(RBC)、血红蛋白含量(Hb)、红细胞脆性(Eof)、红细胞沉降率(ESR)、红细胞比容(Hct)、平均红细胞血红蛋白(MCH)、平均红细胞血红蛋白浓度(MCHC)和平均红细胞体积(MCV)等血液生理指标进行了测定,结果显示,黑斑原鮡与其他鲇形目鱼类相比,具有相似的Hct, Hb、MCH和MCHC,较低的RBC和较高的MCV。对血糖(GLU)、尿素(UREA)、肌酐(CREA)、白蛋白(ALB)、球蛋白(GLB)、白蛋白/球蛋白(A/G)、直接胆红素(DBIL)、总胆红素(TBIL)、总胆固醇(TC)、甘油三酯(TG)、总蛋白(TP)、碱性磷酸酶(ALP)、谷丙转氨酶(ALT)、谷草转氨酶(AST)等血液生化指标的测定结果表明,黑斑原鮡的AST高于其他鲇形目鱼类。9.利用光镜和电镜对黑斑原鮡血细胞形态与结构的研究表明,黑斑原鮡外周血中包括红细胞和五种白细胞：淋巴细胞、嗜中性粒细胞、单核细胞、血栓细胞和浆细胞。与其他鲇形目鱼类相比,黑斑原鮡血细胞的形态和结构具有特殊之处,如较大体积的红细胞、不存在嗜酸性粒细胞和嗜碱粒细胞以及存在各种形态的血栓细胞(裸核、纺锤形、蝌蚪形、卵圆形和聚集到一起的血栓细胞)。更多还原

【Abstract】 Glyptosternum maculatum, one species of Glyptosternoids, is distributed in the mid-reaches of Yarlung Zangbo River as well as its tributaries in the Tibetan Plateau of China. G. maculatum belongs to the genus Glyptosternum (McClelland), the family Sisoridae, in the order Siluriformes. Besides the celiac liver, there is another structure called "exo-celiac liver" located between skin and muscular layer, and connected with the celiac liver by a funiform tissue, "joint belt". According to previous research, "exo-celiac liver" has been proven to be a unique normal tissue in G. maculatum. This dissertation studied the liver morphogenesis of the yolk-sac larvae, the physiological and biochemical differences between celiac liver and exo-celiac liver, the liver characteristics of sisorid catfishes, and the haematology. The main results are shown as follows:1. The location of the liver in G. maculatum first appeared in the left side of primary alimentary tract. The liver morphogenesis was divided into three phases:(1) no exo-celiac liver, (2) appearance of the "protrusion", and (3) appearance of exo-celiac liver. The exo-celiac liver progressively forms as the adjacent tissues grow larger (muscle of parietal peritoneum thickens, the size of bony bladder and oesophagus is enlarged). Exo-celiac liver located in the abdominal cavity just as celiac liver, so we considered that’exo-celiac liver’should be instead of’attaching liver’, and the’celiac liver’should be instead of ’main liver’accordingly.2. The microstructure and ultrastructure of main liver and attaching liver were similar in G. maculatum. In histology, joint belt as the connecting bond of material transport between main liver and attaching liver, its vein, artery and bile duct were larger than both of them.3. We determined the contents of biochemical compositions, enzyme activities and total antioxidant capacity of main liver and attaching liver in G. maculatum. The contents of protein and lipid, total antioxidant capacity, activities of SOD and CAT in main liver were all slightly higher than those in attaching liver. The content of glycogen, activities of LDH, ALT and AST were all lower than those in attaching liver. However, there were no significant differences in all parameters between main liver and attaching liver (P>0.05).4. We obtained the specific sequences ofβ-actin、Cu-Zn SOD、Mn SOD and CAT, and determined the relative gene expression of Cu-Zn SOD, Mn SOD and CAT in main liver and attaching liver of G. maculatum by fluorescence quantitative PCR with P-actin as a control. The relative mRNA levels of Cu-Zn SOD, Mn SOD and CAT in main liver were significant higher than those in attaching liver (P< 0.05).5. Eighteen species (10 genera) of sisorid catfishes were dissected in this research,7 species of which possessed obvious attaching liver, whereas the other 11 species (4 genera) had no attaching liver. The differences of hepatic morphology occurred among different genera. The glyptosternoid fishes possessed attaching liver, whereas Bagarius, Gagata, and Glypothorax had no attaching liver. The hepatic heteromorphosis appeared in different species whthin the same genera. Pseudecheneis sulcatus possessed attaching liver, whereas Pseudecheneis intermedius had no attaching liver. The order of the weight ratio of attaching liver to main liver is P. sulcatus, Pseudexostoma< Pseudexostoma< Euchiloglanis< Glyptosternum< Exostoma< Glaridoglanis.6. The correlation analysis of the tracks of different groups and elevation of Chinese sisorids (significantly positive correlation) showed that as the elevation became higher, the hepatic characteristics of sisorids had a trend as follows:no attaching liver (Bagarius and Gagata)→appearance of the hepatic protrusion(Glypothorax)→evident protrusion (Pseudecheneis intermedius)→appearance of attaching liver (Pseudecheneis sulcatus)→larger attaching liver (Glyptosternini fishes). Attaching liver formed as the body forms of sisorids changing in the process of adapting themselves to the environment of high elevation and torrent stream, which is an acclimation to particular environment.7. Based on the relative size of attaching liver from small to large, the phylogeny of the glyptosternoid fishes in this research presented the following tendency of evolution: Pseudexostoma (attaching liver is obviously smaller than main liver)→Pareuchiloglanis (attaching liver is smaller than main liver, but larger than Pseudexostoma)→Euchiloglanis (attaching liver is smaller than main liver, but larger than Pareuchiloglanis)→Glyptosternum (attaching liver is smaller than main liver, but larger than Euchiloglanis)→Exostoma (attaching liver is similar to main liver)→Glaridoglanis (attaching liver is larger than main liver).8. Red blood cell count (RBC), haemoglobin concentration (Hb), haematocrit (Hct), crythrocyte osmotic fragility (maxEof and minEof), the erythrocyte sedimentation rate (ESR), mean cell volume (MCV), mean cellular haemoglobin content (MCH), and mean cell haemoglobin concentration (MCHC) were determined. Compared with other Siluriformes fishes, G. maculatum showed similar mean values for Hct, Hb, MCH, and MCHC and had slightly lower RBC and higher MCV. Alanine aminotransferase, aspartate aminotransferase (AST), alkaline phosphatase, total protein, albumin, globulin, albumin/globulin ratio, total bilirubin, direct bilirubin, urea, creatinine, glucose, total cholesterol, and triglyceride were assayed. The result showed that the value of AST in G. maculatum was obviously higher than that in other Siluriformes fishes.9. The peripheral blood cells of a sisorid catfish G. maculatum were studied by light microscope and transmission electron microscope. Erythrocytes and five types of leucocytes:lymphocytes, heterophils, monocytes, thrombocytes, and plasma cell were characterized in G.maculatum blood. The morphology and structure of blood cells of G. maculatum were basically similar to those of other Siluriformes fish species, although there were also main differences, such as larger erythrocytes than other catfishes, absence of basophils and acidophils, and various types of thrombocytes (five types:lone nucleus, fusiform, tadpole-like, oval, and in a cluster).更多还原