碩士 / 國立臺灣海洋大學 / 食品科學系 / 101 / The common carp release zinc that stored in the fibroblast of digestive tract tissue when under stress. Free zinc combines with the transferrin in blood, and the zinc-transferrin complex is transported to head kidney by blood vessels. Zinc-transferrin complex interacts with the transferrin receptor on head kidney cells and were internalized to the cell, which stimulates the proliferation of erythroid cell to against stress in common carp. To understand whether zinc stimulates the proliferation of erythroid cell in the common carp head kidney is universal or not, grass carp and bighead carp which also are Cyprinidae, and tilapia which is classified to be Cichlidae, were used to study the effects of zinc on the proliferation of the erythroid cells in their head kidney.
The grass carp, bighead carp, and tilapia head kidney cells were suspension-cultured with 4 different medium: medium only, medium + 10 % grass carp serum / bighead carp serum / tilapia serum, medium + 0.3 mM ZnCl2, medium + 10 % grass carp serum / bighead carp serum / tilapia serum + 0.3 mM ZnCl2 for 0, 1, 2, 4 days. The cell levels in total cell, and fractions 1 and 2 cell after Percoll separation were measured. The results show grass carp head kidney fraction 1 cell concentration that cultured with serum and ZnCl2 proliferated 6.22 times higher than the start after 4 days. Similar to grass carp, bighead carp head kidney fraction 1 cell level that cultured with serum and ZnCl2 were 3.02 times higher than the start after 4 days. But tilapia head kidney fraction 1 cells cultured with tilapia serum, regardless with or without ZnCl2, both proliferated to 2.89 and 2.82 times after 4 days. The results showed that adding ZnCl2 is no useful to tilapia head kidney fraction 1 cell growth.
The grass carp, bighead carp, and tilapia head kidney cells were suspension-cultured with serum and ZnCl2 for 4 days, and then extracted with lubrol solution. The detergent extraction was subjected to Zn2+-IMAC (Immobilized metal ion affinity chromatography). The results show after 4 days, the Zn-binding proteins in head kidney of grass carp, bighead carp, and tilapia all increased 2-3 times higher than day 0. SDS-PAGE and nano-LC-MS/MS analysis indicated that all the Zn-binding protein were transferrin variants.
These results indicated that the common carp, grass carp, bighead carp, and tilapia head kidney cells all have the same mechanism. In vitro, zinc combines with transferrin, and then the zinc-transferrin interacts with the transferrin receptor on head kidney cell. The zinc-transferrin complex is transported into the cell by endocytosis. In this case, zinc is the mitogen, and stimulates the proliferation of erythroid cells. After 4 days culture, zinc-transferrin induced common carp and grass carp head kidney cells proliferated 4.7 and 6.2 times, which were higher than bighead carp being proliferated only 3.6 times. The effect of zinc on tilapia head kidney cell growth is the least, only 2.8 times. However, in vivo, there are no enough zinc concentration in the grass carp and bighead carp head kidney cells, stimulation of the cell by zinc are limited. On the contrary , because the common carp has high zinc concentration in their digestive tracts, they can endure high stress.
Common carp serum were used to replace grass carp / bighead carp serum in culturing grass carp / bighead carp head kidney cells. The results show that the fraction 1 cells also proliferation 5.28 and 3.97 times higher after 4 days. The results indicate that common carp serum has similar growth effect as their own serum. However, when common carp serum was used to replace tilapia serum, the tilapia head kidney fraction 1 cells only increased 1.9 times, which is lower than its own serum. The possible reason is Cyprinidae fish and tilapia transferrins have been diverged through evolution, so the common carp serum lost the ability to stimulates tilapia head kidney cell proliferation. In addition, the human-transferrin could not stimulate the proliferation of grass carp, bighead carp, and tilapia head kidney cells. This might be their homology between human-transferrin and grass carp, bighead carp and tilapia transferrins are too low to act on their head kidney cells.
When the stressed grass carp and tilapia head kidney cells were cultured with serum and ZnCl2 for 4 days, the head kidney fraction 1 cells did not proliferate. The head kidney cell composition of the resting and stressed grass carp, and tilapia were compared. It was found that the fraction 1 cell concentrations of stressed fishs are higher than that of resting fishs. These results indicated that stressed grass carp and tilapia head kidney cells could not proliferate new erythroid cells when cultured with serum and ZnCl2 in vitro.
| Identifer | oai:union.ndltd.org:TW/101NTOU5253033 |
| Date | January 2013 |
| Creators | Jhe-Ruei Shiu, 許哲睿 |
| Contributors | Sen-Shyong Jeng, 鄭森雄 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 44 |
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