【作者】 刘奕；

【导师】 范兆廷；

【作者基本信息】 东北农业大学 ， 水生生物学， 2010， 硕士

【摘要】 增殖细胞核抗原(PCNA)是仅存在于增殖细胞的细胞核中的一类蛋白质,通常被用作细胞增殖的标记物。通过对虹鳟外周血及造血器官中PCNA表达的定位研究,可以确定外周血红细胞及造血器官中细胞的增殖能力,从而为鱼类多倍体红细胞研究提供基础资料。本试验观察并对比了二、三倍体虹鳟Oncorhychus mykiss(Walbaum)外周血红细胞的形态结构的差异;运用免疫细胞化学方法、免疫组织化学方法检测外周血红细胞中是否存在增殖细胞核抗原(PCNA)的表达并对其进行了定位研究。运用流式细胞术对外周血红细胞PCNA的表达量进行了定量研究;检测了二三倍体虹鳟外周血中S期红细胞所占比例;对造血器官中PCNA的表达进行了定位研究,确定了造血组织的分布及各器官造血的活跃程度。试验结果表明:1.三倍体虹鳟外周血红细胞与二倍体相比较体积较大,细胞形态更为细长。三倍体红细胞及其细胞核的体积分别为二倍体的1.49倍和1.50倍,与理论值1.50倍相符合。在三倍体虹鳟外周血中可见一定比例的畸形红细胞,可以大概分为缢痕型、哑铃型和不规则型。这些畸形细胞在二倍体中也有少量存在,但比例极小。二、三倍体中畸形细胞的比例分别为0.08%和8.5%。在二、三倍体的外周血液中还可见一定比例的未成熟红细胞,分别占红细胞总数的8%和11.9%,二、三倍体之间差异极显著。2.通过光学显微镜观察免疫组织化学染色后二、三倍体的红细胞中有棕黄色沉淀附着的PCNA阳性染色细胞占整个红系细胞总数的0.2%和6.4%(试验所用抗体为小鼠抗PCNA单克隆抗体(克隆号:PC10))。阳性染色细胞的形态与未成熟红细胞的形态一致或相近。三倍体中呈PCNA阳性的细胞约占未成熟红细胞总数的57.9%。而二倍体中PCNA阳性细胞数量极少,仅占未成熟红细胞数的2.5%。3.流式细胞仪检测二、三倍体外周血液中红细胞处于S期的细胞比例分别为0.68%和4.05%,差异极显著。通过荧光抗体标记并进行流式细胞仪检测得到二、三倍体中有PCNA阳性表达的红细胞比例分别为0.37%和8.39%,差异极显著。4.抗PCNA单克隆抗体(PC10)可以与细胞中增殖细胞核抗原特异性结合,通过蛋白质电泳检测出一条由免疫反应产生的35-36 kDa的蛋白质条带。并且可以看出,抗PCNA单克隆抗体(PC10)可以与大鼠脾脏,以及虹鳟肝脏、肾脏和三倍体外周血细胞发生交叉反应,其可用于对虹鳟增殖细胞核抗原的检测。5.采用TUNEL法检测虹鳟外周血中的凋亡细胞,无论二倍体还是三倍体的外周血中均有呈棕黄色染色的凋亡细胞,但这些细胞属于血液中自然凋亡细胞。畸形红细胞在TUNEL检测中并未表现出凋亡细胞所应有的棕黄色沉淀,所以它们不是凋亡细胞。从而排除了畸形细胞产生的一种可能性即细胞凋亡。6.检测虹鳟造血器官中PCNA的表达可知,头肾中大量造血祖细胞集中分布于血窦内并且呈较强的PCNA阳性。造血祖细胞在头肾组织中也有广泛分布,并且增殖活跃。在肾脏中,PCNA阳性细胞均匀分布于肾小管和集合管之间,均为造血细胞。同时小管内皮细胞也有阳性染色。脾脏网状结构内有大量血细胞分布,其中有部分细胞呈现PCNA阳性染色为造血祖细胞。肝脏中有少量肝细胞也表现PCNA阳性染色,而肝细胞中可见双核或哑铃形细胞;肝脏中的造血组织主要分布于肝血窦及被膜下区域,PCNA免疫组织化学反应后可观察到阳性染色。更多还原

【Abstract】 Proliferating cell nuclear antigen is a kind of protein which only can be found in the nuclei of proliferating cells. It was used as a marker of the proliferation of cells. The located research of the expression of PCNA in peripheral blood and hematogenic organs in rainbow trout could determine the reproductive activity of erythrocytes, and offer basis data for the research of erythrocytes in polyploidy fish.In this paper, the morphology and construction of erythrocytes in peripheral blood of di- and triploid rainbow trout Oncorhychus mykiss(Walbaum)was observed and contrasted. The expression of PCNA in erythrocytes was examined by the method of immunohistochemistry and flow cytometry. And the proportion of the erythrocytes in S phase was detected at the same time. Positioning study was took place in the hematogenic organ to determine the distribution and active degree of the hemopoietic tissues. The results were as follows:1. Contrast with that of diploids, the erythrocytes in triploid rainbow trout was larger and more slender. The volume of the erythrocytes and the nuclei of the erythrocytes in triploid were 1.49 and 1.5 times larger than that of diploids which were corresponded to the expectant theoretical value of 1.5. In peripheral blood of triploid rainbow trout, about 8.5% of erythrocytes nuclei showed abnormal shapes of constriction, dumbbell, and irregular shape. This phenomenon could also be seen in diploids but the proportion was very small which was 0.08%. Immature erythrocytes could also be seen in the peripheral blood of di- and triploid rainbow, and the proportions of which were 8% and 11.9% which had significant difference.2. Brown precipitates could be observed by light microscope in the nuclei of PCNA positive cells after staining by the method of immunohistochemisty. The rates of PCNA positive erythrocytes in the peripheral blood of di- and triploid rainbow were 0.2% and 6.4% in erythroid cells (antibody used in the experiment was anti-PCNA monoclonal antibody (PC10)). The shapes of positive erythrocytes corresponded to immature erythrocyte. The proportions of positive erythrocytes in immature erythrocytes of di- and triploids were 2.5% and 57.9%.3. Determining by flow cytometry, the rate of the erythrocytes in peripheral blood which were under S phase was 0.69% in diploids and 4.05% in triploids and the proportion of antibody labeling cells was 0.37% in diploids and 8.39 in triploids.4. Anti-PCNA monoclonal antibody (PC10) could specifically bind with PCNA in proliferative cells which could be detected by protein electrophoresis which showed a 35-36 kDa protein strap. An immunoreactive polypeptide with an electrophoretic mobility is found in rainbow trout liver, spleen and the peripheral blood cells of triploids. The staining intensity demonstrates that peripheral blood cells of triploids whose cells possess a lower proliferatory capacity.5. TUNEL, which is a method of apoptosis detection was used to determine the abnormal erythrocytes in peripheral blood of di- and triploid rainbow trout. The apoptotic erythroid cells were indicated by the brown reaction products. The result showed that apoptosis erythrocytes could be found both in di- and triploid rainbow trout, and these cells were normal apoptosis erythrocytes. Abnormal erythrocytes showed negative to TUNEL probing, so they were not the cells which undergone a procession of apoptosis. Thus the presumption about abnormal erythrocytes of apoptosis could be eliminated.6. The head kidney of rainbow trout was enriched in hematopoietic tissue but lack of the structures related with excretory system, and the hematopoietic cells proliferated actively which showed that it was one of the most important hematopoietic organs in rainbow trout. In this study, PCNA positive cells in kidney were also centered to the endothelial cells which may suggest that there were interactions between hematopoietic endothelial and renal tubular cells. In spleen strong immunoreactivity is observed, and PCNA is expressed exclusively in the hematopoietic compartment. The positive cells are hematopoietic progenitor cell. A small quantity of hepatic cells was positive staining which dispersed mainly in hepatic sinusoid and the region of subvelamen.更多还原