碩士 / 東吳大學 / 微生物學系 / 86 / In the crustaceans, the nonspecific immune response is the primary defense system and the cellular immunity is more important. To study and clarify the roles of different haemocytes in cellular immune responses, separation and classification of haemocytes are essential. Four monoclonal antibodies, including Z5E10、Z6A5、Z6A6 and Z6H8, developed in our laboratory are specific binding to the different haemocyte types of tiger shrimp (Penaeus monodon), respectively.In this study, the four MAbs were used to characterize and separate the different subpopulations of haemocytes, and further to detect the expression of some intracellular defense enzymes.By using the immunogold and immunofluoresence stain, we found that all four MAbs can recognize a few hyaline cells (HCs) and the most of semigranular cells (SGCs) and granular cells (GCs). The results from double-MAb immunofluoresence stain showed, except that haemocytes recognized by Z5E10 and Z6H8 were identical, that there were four conditions via double-MAb recognition, such that one group of haemocytes can be recognized by both Z5E10 and Z6A6, one can not berecognized by the both MAbs, one was recognized by Z5E10 alone, and the other one was recognized by Z6A6 alone. Therefore, we suggest that there are at least 6 subpopulations of haemocytes from tiger shrimp.Using beads conjugated with concanavalin A (ConA) or one of the four MAbs to separate haemocytes into the different subpopulations via positive , negative and double-negative selections, the results showed that the haemocyte sample of HC can be obtained via the three selections, and the percentages of HC in the haemocyte sample were 82% ~ 90% for positive selection, and were more than 90% for either negative or double-negative selection. However, neither SGC sample nor GC sample can be obtained by the all three selections. In addition, the results from the expressions of a variety of defense enzymes revealed that both of β-glucuronidase and α-naphthyl acetate esterase widely existed in haemocytes which can or can not be recognized by the four MAbs; however, acid phosphatase was only detected in the haemocytes which can not be recognized by all MAbs. Similarly, the expression of prophenoloxidase primarily appeared in the haemocytes which can not be recognized by MAbs except Z6A6. Finally, although inducible nitric oxide synthase (iNOS) can not be found by immunfluoresence stain, the presence of iNOS within shrimp haemocytes can be detected by using Western blotting.In conclusion, haemocytes from tiger shrimp can be classified to at least 6 subpopulations via the specific recognization of the four MAbs used in this study; as well as, HC sample can be collected via negative selection, but either SGC nor GC sample can. We consider that the four MAbs plus ConA are not enough to separate the haemocytes into different subpopoulation. Therefore, it is essential to develop more MAbs for studying in defense roles of different subpopulations of haemocytes.
| Identifer | oai:union.ndltd.org:TW/086SCU00381008 |
| Date | January 1998 |
| Creators | Sun Ryan, 孫咸義 |
| Contributors | Sung Hong-Hong, 宋宏紅 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 0 |
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