姜延年Ju, Yu-Ten;Jiang, Yan-Nian臺灣大學:動物科學技術學研究所穆姵璇Mu, Pei-HsuanPei-HsuanMu2010-05-112018-06-292010-05-112018-06-292009U0001-1708200913535400http://ntur.lib.ntu.edu.tw//handle/246246/182076在成年的哺乳動物，乳腺發育會經歷未懷孕、懷孕、泌乳，及回復等四個時期，在這四個時期的輪換，乳腺歷經多重的增殖、分泌乳汁，及退化。利用cDNA雜合扣除法發現Rad21 mRNA在小鼠退乳與泌乳時期表現量差異大，以西方吸漬法檢測基因表現量時發現，全長的Rad21大量表現於小鼠懷孕及泌乳期乳腺中，當乳腺開始進入離乳期時，全長Rad21表現量逐漸降低，然而在山羊乳腺的Rad21仍屬未知，利用不朽化山羊乳腺上皮細胞做為基因轉殖動物蛋白質表現之測試平台，故本研究之目的在於瞭解Rad21於山羊乳腺細胞內所扮演之角色，並探討於山羊乳腺發育時期的基因調控機制。 Rad21是一種核蛋白，為連接姐妹染色分體之cohesin主要次單位，當細胞進行有絲分裂至anaphase時，Rad21會被酵素截切，以分離姐妹染色體。此外，研究發現Rad21也與引發細胞凋亡有關，當外在誘發細胞凋亡時，不同於有絲分裂酵素截切的位置，Rad21亦會被截切成N’端及C’端兩片段，其中某一片段會由細胞核運行至細胞質，進而引起細胞凋亡。羊之Rad21基因與人類、小鼠、牛之Rad21基因其蛋白質序列具有高度保留性，從山羊初期培養乳腺上皮細胞中選殖出山羊之全長Rad21基因。為進一步的確認在發育乳腺中全長Rad21及其兩種蛋白酶截切裂解產物與caspase-3間的相關性，於山羊乳腺上皮細胞 (CMEC) 中分別大量表現全長Rad21、N端與C端Rad21重組蛋白，以誘導細胞凋亡的藥物etoposide處理36小時後，經由西方吸漬法證實當外源性表現的全長Rad21、 N端與C端Rad21後，並未提早出現活化態caspase-3片段，代表無論全長Rad21或是其裂解產物不會誘導細胞凋亡。此外，全長的Rad21也未發現被截切所產生一相似於C端Rad21裂解產物大小的片段。為進一步了解外源性表現的全長Rad21、 N端與C端Rad21是否會影響細胞凋亡的過程，利用Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay偵測細胞凋亡，以etoposide處理36小時後，經螢光免疫染色發現外源表現全長goat Rad21、C端或N端Rad21的細胞，其死亡率顯著低於沒有外源表現的細胞( p<0.05, n=3)，故推測全長Rad21、C端或N端Rad21有抑制細胞凋亡的作用。知細胞存活主要路徑為PI3K-Akt，為了證實Rad21是經由活化此路徑而抑制細胞凋亡，分別同時表現全長Rad21、N端與C端Rad21與wild-type Akt重組蛋白於山羊乳腺上皮細胞 (CMEC) 中，以etoposide處理36小時後，經由西方吸漬法證實表現全長Rad21、N端與C端Rad21組別的磷酸化Akt(p-Akt)表現顯著增加，而存活下來的細胞幾乎能表現外源基因，證實無論全長Rad21、N端或C端Rad21都可以增加Akt磷酸化，透過活化PI3K-Akt路徑達到抑制細胞凋亡的功能。 綜上所述，全長Rad21蛋白在泌乳時期維持大量表現，當進入離乳初期細胞進行大規模凋亡時，表現量卻下降。由試驗證實，全長Rad21有抑制細胞凋亡的功能，推測Rad21可以維持乳腺泌乳時期之細胞數量，且避免細胞進行凋亡而提早進入離乳。Mammary epithelial cells periodically undergo proliferation, differentiation, growth arrest, and apoptosis during each pregnancy-weaning cycle. Using PCR-select cDNA subtraction strategy, we found that Rad21 mRNA has highly diversity of expression between involution and lactation stage of the murine mammary gland. To begin with, we examined the expression pattern of Rad21 in different developmental stages of mammary gland to establish its correlative function in mammary glands. The Rad21 protein expression profile was demonstrate with western blotting technique. The result showed that Rad21 was highly expressed in pregnancy and lactation stage but low in the beginning of weaning. However, Rad21 was still unclear in goat mammary gland development. We used immortal caparin mammary epithelial cell (CMEC) as a model to study the role of Rad21 and its regulatory mechanism in development of goat mammary gland. Rad21 is a nuclear protein, and Rad21 is one of the major cohesin subunits that holds sister chromatids together until anaphase, when proteolytic cleavage by separase, allows chromosomal separation. In addition, the cleavage of Rad21 also occurs during apoptosis induced by diverse stimuli. This apoptotic cleavage site is distinct from previously described mitotic cleavage sites. Rad21 is cleaved in carboxy-terminal and amino-terminal products. One of cleaved products is translocated to the cytoplasm early in apoptosis before chromatin condensation and nuclear fragmentation. According to goat Rad21 has highly conservation with human, murine and bovine, we cloned goat full-length Rad21 (gRad21) from primary cultured goat mammary gland. To investigate the effect of Rad21 and its cleavage products to the activation of caspase-3 in CMEC, we transiently transfected full length, N-and C-terminal Rad21 into CMEC and treated with etoposide for 36 hours, then observed actived caspase-3 by western blotting. It showed no effect on actived caspase-3 expression profile, suggested that no matter exogenous full length, N and C-terminal Rad21 did not induce apoptosis. Further more, full length Rad21 did not be cleaved into N-and C-terminal products. Using Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to investigate apoptosis, after eioposide treatment 36 hours, we found that it was a small amount of cell expressed Rad21 and apoptosis at the same time. We speculated full length, N- and C-terminal Rad21 can inhibit apoptosis. Combined together, these results show that, full length Rad21 protein remain high expression during lactation but reduce expression as entry of weaning. These data hind that expression of full length and C-terminal Rad21 may have a role in inhibition promoting apoptosis of mammary epithelial cell to remain mammary gland lactation and afford early entry of weaning.目錄 頁次錄 ..3次 ...4、前言 ...5、中文摘要 ...6、英文摘要 ...8、文獻檢討 .10、材料與方法 ……..20、結果 ......28、討論 ….33、結論 ….38、參考文獻 ….39、附錄 ….56application/pdf941895 bytesapplication/pdfen-USRad21乳腺凋亡mammary glandapoptosisthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182076/1/ntu-98-R96626004-1.pdf