錦鯉細胞株之熱休克蛋白質表現

Abstract

本論文首先利用源自錦鯉精巢、鰓和鰭組織而來的細胞株為材料，配合SDS-PAGE膠體電泳，及自動放射顯影技術，瞭解熱休克蛋白質在CCT、CCG和CCF細胞株內的合成表現。實驗結果顯示，在37℃時，CCT細胞可合成全部的6種熱休克蛋白質（hsp97、87、72、68、30和27），而CCG和CCF細胞則只可合成hsp97、87和70等3種，其必須在40℃時方可合成出所有的5種熱休克蛋白質（hsp97、87、70、33和27）。其中以hsp70的合成速率最快也最明顯，hsp27則最慢亦不明顯。當在持續受熱環境下5-7小時後，hsp72或hsp70的合成速率明顯降低，hsp33或hsp30更停止生產，hsp87雖有下降但不明顯，而hsp27則繼續合成至更長的一段時間後方下降。 接著自SDS-PAGE中純化出熱休克蛋白質，並以之製備出兔抗熱休克蛋白質抗體，再以免疫轉印和螢光顯微技術觀察錦鯉3細胞株在被持續高溫（37°或40℃）緊迫時，其內熱休克蛋白質量及位置的動力改變。發現hsp87大量存在未受熱緊迫的細胞內，其在細胞內的總量必須在受熱12小時後才會明顯增加，然不論受熱前或受熱後其分佈位置均明顯的座落在細胞質上，然若改以免抗CCG hsp87抗體處理時，其抗hsp87螢光則又出現在整個細胞上但以細胞核為主。Hsp70則在細胞受熱後立即大量的生產且依序自細胞質進入核仁、細胞核並推積至細胞核四周的細胞質。Hsp33在細胞受熱後明顯大量的增加，其先大量出現在細胞核內，然後轉移至細胞質，但會在受熱12到24小時之間退化消失。Hsp27在常溫即存在著，細胞受熱6小時後到達最大量，並以此定量保持於細胞內。其在常溫時約有半數實驗細胞以明顯的抗hsp27螢光表現在細胞核內，細胞受熱後此位於細胞核的hsp27會逐漸消失，整個細胞也少有hsp27的存在。但隨著加熱時間的遞增，抗hsp27螢光會以絲狀出現在細胞核四周，最後表現在整個細胞質。 當以免疫轉印法探討各個熱休克蛋白質在18種魚類細胞株間的抗原性關係和保存性時，發現兔抗hsp87和hsp70抗體，不論抗原是來自CCT或CCG細胞，均對所有18種魚類細胞株有反應，而兔抗CCT hsp30抗體只和6種細胞株反應，兔抗CCG hsp33抗體更少，只和CCG和CCF細胞反應。兔抗CCT hsp27抗體也只和8種細胞反應。這些結果顯示熱休克蛋白質在18種魚類細胞株中，以hsp70的保存性最高，而hsp87次之，hsp27又次之，hsp33最差。 最後，以螢光抗體法探討熱休克蛋白質和細胞週期的關聯時，則發現未受緊迫的G1期CCT細胞，其抗hsp72螢光以成串小點狀顆粒表現在細胞質，抗hsp30螢光則很微弱。當以螢光顯微鏡觀察未受緊迫的同步化細胞，發現其抗hsp70和30的螢光並不會因細胞週期的運轉而不同。將各期細胞加熱則均會產生熱休克蛋白質，同時也和未同步化的細胞一樣，有很多的細胞在受熱緊迫後仍如對照組一般。這些結果證實，有些細胞在受緊迫後仍未出現抗hsp72的螢光，其原因應和細胞週期無關。

The exposure of three color carp cell lines (CCT, CCG and CCF) to an elevated temperature resulted in the over-production of heat shock proteins. The kinetics of their syntheses at 37°, 40° or 43℃ were studied. With various heat shock treatments, the synthesis of hsp70 was the most rapid and prominent, and hsp27 was the slowest. Prolonged heat shock at 40℃ beyond 6 h resulted in decreased syntheses of hsp87 and 70, and a synthesis of hsp33 or hsp30 ceased, but a synthesis of hsp27 continued. Heat shock proteins in CCT and CCG were purified, and rabbit antibodies were raised against them. These antibodies were used in immunoblot and immunostain to detect the dynamic changes in the quantity and intracellular distribution of the three heat shock proteins in color carp cell lines stressed by heat shock at 37℃ or 40℃. In CCF and CCG cells, hsp87 was enhanced immediately after heat shock; however, the cellular hsp87 did not increase to a relatively elevated level which was detected evidently by western immunoblot until 6 h heat shock. Through immunofluorescence microscopy, it was observed to localize in the cytoplasm before and after heat shock. During heat shock, significant amounts of hsp70 were immediately synthesized and moved successively from the cytoplasm to the nucleolus and the nucleus and then overflowed into to the perinuclear region. In addition, the intracellular level of hsp33 increased significantly as quickly as hsp70 after heat shock but disappeared completely 6-12 h following heat shock. Furthermore, during heat shock, hsp33 mainly appeared first at the nucleus; then it was found exclusively in the cytoplasm and finally disappeared from the cytoplasm after 6-12 h. The level of hsp27 declined immediately after heat shock then began to synthesize progressively. After reaching its maximal level, a steady-state level was maintained for the remainder of the experimental period. Immunofluorescence microscopically, hsp27 could be detected in the nucleus of about half of the tested unstressed CCF cells. During heat shock, the cells with nuclear staining appeared diminished and little or no fluorescence was observed in the CCF cells. Subsequently, filament staining within the perinucleus region began to increase and then intense hsp27 cytoplasmic staining appeared. CCF cells were heated at 40℃ for 10 h then transferred to 31℃. Immunofluorescent stains show that the level of hsp87, hsp70, and hsp33 returned to the preheat-shock levels after 36 h of recovery. However, intense hsp27 cytoplasmic staining remained even after a 36-h recovery period. These results suggest hsps is degraded in the cytoplasm. Similar results were observed from CCT cells. After heat shock, hsp87 mainly appeared in the cytoplasm, hsp72 and 30 in the nucleus. However, when cells were restressed at 37℃ for 8 h after a recovery period, we found the intensity and localization of the three anti-CCT hsps staining changed from those following an initial stress for 8 h. These data suggested that the synthesis of the hsps was self-regulated. These antibodies were also used to detect antigenic material of 18 fish cell lines derived from widely different species. We found that anti-hsp87 and 70 but not 33, 30 or 27 antibodies cross-reacted with proteins of similar molecular weights in all tested cell lines. These data provided further evidence for the conservation of high molecular weight hsps.