臺灣博碩士論文加值系統

緊迫鯉魚之血液其細胞濃度與鋅濃度皆高於靜養鯉魚，而緊迫
鯉魚頭腎之細胞濃度與鋅濃度也皆高於靜養鯉魚。由於頭腎係鯉魚之造血器官，在緊迫條件下，鯉魚血液細胞濃度之會增高，可能係由
頭腎製造而來。為知鯉魚頭腎在緊迫狀態下，如何增生細胞，此種
增生與鋅之關係，乃自市場購買鯉魚 (緊迫鯉魚) 及在實驗室靜養鯉魚 (靜養鯉魚)。以Percoll 密度梯度 (ρ為1.02 g/ml 及1.07 g/ml) 分離15尾靜養鯉魚、14尾緊迫鯉魚之頭腎組織中的細胞，將其分為兩層，L1及L2細胞，並測定其細胞濃度，結果發現，緊迫鯉魚之L1細胞之
細胞濃度顯著地高於靜養鯉魚之細胞濃度，其值為523 x 106 cells/g tissue比118 x 106 cells/g tissue。而L2的細胞濃度分別為746 x 106 cells/g tissue比776 x 106 cells/g tissue，其間並無顯著差異。在L1細胞中，
緊迫鯉魚增生之細胞主要為大小約為6 μm之細胞，其量為354 x 106 cells/g tissue而靜養鯉魚只有86 x 106 cells/g tissue，其間相差4倍。
由Giemsa 染色結果中發現，鯉魚頭腎組織中的L1細胞，主要為外觀呈現圓形，大小約為6 μm之細胞。此類細胞具有薄層的細胞質，及圓形的細胞核，其外觀特點相當類似造血幹細胞/前驅細胞。另外，由43kDa鯉魚鋅結合蛋白質免疫螢光染色的結果得知，緊迫鯉魚之L1細胞有較多之具有螢光反應之~ 6 μm之細胞，及小光點在 ~ 6 μm之
細胞之細胞膜上。在靜養鯉魚，頭腎細胞中之90%皆位於L2。Giemsa 染色指出其中含有多種類之細胞，以o-Dianisidine染色後，發現在L2層，緊迫鯉魚有較多之紅血球。
將鯉魚頭腎組織中的細胞，以4種不同培養基：只有培養基，
培養基 + 10 % 鯉魚血清 ， 培養基 + 10 % 鯉魚血清 + 0.3 mM 氯化鋅及培養基 + 0.3 mM 氯化鋅進行系列繼代培養 (serial subculture) d0-d3後，再將培養之細胞以Percoll 密度梯度分離出L1及L2細胞，測定整體細胞、L1及L2的細胞濃度，並製作其生長曲線，結果發現，以添加氯化鋅及鯉魚血清組生長最好。增生之細胞主要在L1層之
細胞。鋅會誘導鯉魚頭腎細胞之增生，但必需同時添加鯉魚血清。
為知鋅對鯉魚頭腎細胞增生之功能，乃在含有鯉魚血清之培養基中，分別添加氯化鋅 (0.3 mM) 及未添加鋅，在固定時間 (h0-h96)
培養細胞，再以Percoll 密度梯度分層細胞，結果發現，同時添加氯化鋅及鯉魚血清，可使L1細胞快速增生，細胞由h0之3.60 x 106 cells/ml增生至h96之12.5 x 106 cells/ml。添加氯化鋅及鯉魚血清組L1細胞之細胞倍增時間為6h；僅添加血清組為19h。鋅可相當程度加速細胞之增生。
將添加氯化鋅及血清組培養3天後之L1細胞，以免疫螢光染色
發現~ 6 μm 之細胞具有螢光反應，同時，有螢光光點存在於 ~ 6 μm之細胞上，除此之外，培養3d後之細胞，在L1細胞中增加了部份紅
血球，在L2層增生了許多紅血球，僅添加鯉魚血清組，則紅血球較少。氯化鋅除了使L1細胞增生之外，似乎也會促進紅血球之分化。
鯉魚受到緊迫時，鋅會由消化道組織釋出，經由血液，輸往頭腎。鋅很可能係由血液中之攜鐵蛋白 (transferrin) 結合，到達頭腎後，此鋅-攜鐵蛋白之複合物，可能再與頭腎組織中之造血幹細胞/前驅細胞之攜鐵蛋白受體 (transferrin receptor) 結合，進而誘導紅血球之增生，以
協助鯉魚克服緊迫。

The cell and zinc levels in the blood of stressed carp are higher than those of resting carp, at the same time, the cell and zinc levels in head kidney of stressed carp are also higher than those of resting carp. Head kidney is the hematopoietic organ of common carp, the increases of cell level in the blood under stress, may be produced by head kidney. In order to understand how the cells in head kidney of carp proliferate, and the relationship between proliferation and zinc, market carp (stressed carp) and laboratory carp (resting carp) were used as materials. The cells in head kidney of 15 resting carps and 14 stressed carps were separated by Percoll density into 2 layers, layer 1 (ρ = 1.02) and 2 (ρ = 1.07), respectively, and the cell levels in different layers were measured. It was found that the cell levels in layer 1 of stressed carp is significantly higher than those in resting carp, being 532x106 cells/g tissue to 118x106 cells/g tissue. The cell levels in layer 2 were 746x106 cells/g tissue to 776x106 cells/g tissue, respectively, there is no significant difference between the stressed and resting carp. In layer 1, the main proliferated cells had a cell size of ~ 6 μm, for the stressed carp being 354x106 cells/g tissue, and for resting carp being 86x106 cells/g tissue, the difference was 4 times.
In light-microscopic observation of the Giemsa stain, most of the cells in layer 1 were round in size about 6 μm. They have thin layer cytoplasm and a round nucleus, it is very similar to hematopoietic stem cell/progenitor. The immunofluorescent stain with an antibody against the 43-kDa zinc-binding protein indicates there is more immunofluorescent response in the cells in layer 1 of the stressed carp. Ther results of staining with o-Dianisidine indicate there are more RBC in layer 2 of stressed carp.
The cells of common carp head kidney were serial subcultured with 4 types of medium: medium (DMEM/F12) only, medium + 10 % carp serum, medium + 10 % carp serum + 0.3mM ZnCl2, and medium + 0.3mM ZnCl2. At different time intervals, the cultured cells were separated into 2 layers by Percoll density, layer 1 (ρ = 1.02) and layer 2 (ρ = 1.07), respectivey; and the cell level was measured. It was found that the group of medium + 10% carp serum + 0.3mM ZnCl2 has the best growth. Zinc induced the cells in carp head kidney to proliferate, but needed the supplementation of carp serum.
In order to know the growth curve of the cell of carp head kidney under supplementation with or without zinc, the cells of carp head kidney were cultured with carp serum and zinc or carp serum only from h0 to h96. The cultured cells were then separated by Percoll density. The results indicate the cell levels in the group supplemented with carp serum and zinc of layer 1 at h0 was 3.60x106 cells/ml and at h96, 12.5 x106 cells/ml. The population doubling time of cells in layer 1 supplementaed carp serum and zinc was only 6 h, compared to that supplemented with serum only being 19 h. The layer 1 cells after being cultured for 3 days with zinc and carp serum were staind with an antibody against the 43-kDa zinc-binding protein. The results indicate that the 6 μm cells have immunofluorescent response. Besides, there are more RBC cells in layer 2. Zinc not only induce layer 1 cells to proliferate, but may also promote cell to differentiate to RBC.
When carps are under stress, zinc is released from its digestive tract tissue to blood, then transported to head kidney. Zinc is possibly bound to transferrin in blood. In the head kidney, zinc-transferrin complex may be bound to transferrin receptor on hematopoietic stem cell/progenitor cells of the head kidney. This may further the cells proliferate to become RBC to help the carps to overcome stress.

全文摘要.......................................................................................................I
第一章 研究背景與目的 1
第一節 研究背景 1
第二節 研究目的 5
第二章 文獻整理 6
一、造血作用 6
二、側群細胞 (Side Populations, SP) 7
三、硬骨魚的頭腎 8
四、造血幹細胞確實存在硬骨魚腎臟中 9
五、鯉魚的高鋅現象 11
六、鯉魚消化道組織之「鋅結合蛋白質」 11
七、緊迫對鯉魚之消化道組織及造血組織之鋅濃度之影響 12
八、靜養狀態下與緊迫環境中鯉魚血液與造血組織之變化 13
九、Percoll column 分離細胞 13
十、細胞增殖 (Cell proliferation) 14
十一、細胞增殖之控制 (Control of cell proliferation) 14
十二、群體倍增時間 (Population doubling time) 15
十三、細胞分化 (Differentiation) 15
十四、細胞於懸浮 (Suspension) 狀態之生長 16
十五、Giemsa 染色原理 16
十六、o-Dianisidine (3, 3’ diiethoxybenzidine) .................................17
第三章 靜養鯉魚與緊迫鯉魚頭腎組織中 21
細胞組成之不同 21
第一節 前言 21
第二節 材料與方法 22
第三節 實驗結果 28
第四節 討論 29
第四章 靜養鯉魚與緊迫鯉魚頭腎組織中 30
細胞型態之觀察 30
第一節 前言 30
第二節 材料與方法 31
第三節 實驗結果 34
第四節 討論 36
第五章 鋅會誘導頭腎組織中的細胞增生 38
第一節 前言 38
第二節 材料與方法 39
第三節 實驗結果 46
第四節 討論 51
第六章 綜合討論 54
第一節 鋅會刺激鯉魚頭腎組織中的細胞增生 54
第二節 靜養與緊迫狀態下，鯉魚頭腎組織鋅結合蛋白質............55
之不同 55
第三節 增生之細胞為造血幹細胞/前驅細胞 55
表.................................................................................................................58
圖.................................................................................................................66
參考文獻.....................................................................................................78

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