朱有田臺灣大學：動物科學技術學研究所黃瀞瑩Huang, Ching-YingChing-YingHuang2007-11-272018-06-292007-11-272018-06-292006http://ntur.lib.ntu.edu.tw//handle/246246/56622為了解Rad21在正常小鼠乳腺發育表現情形，與其N端與C端裂解產物是否會表現於不同發育階段乳腺中，本研究製備了具專一辨認N端或C端Rad21蛋白質之多株抗體。經由點墨吸漬法，證實了所製備出的抗N端與C端抗體與其抗原做結合能力皆具高感受性，抗體力價皆可達辨認1 pg抗原的能力。亦以免疫細胞螢光染色法證實了產製出的抗N端與C端抗體皆能辨認外源性Rad21與其N端或C端的重組蛋白。以西方吸漬法也證實能辨認到小鼠乳腺組織內源性的Rad21裂解產物。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，Rad21的N端與C端裂解產物表現量則在泌乳末期至離乳初期達到高峯，而後才隨離乳時間增加表現量逐漸下降。此一表現量變化，可能暗示著Rad21在乳腺發育過程扮演不同角色。為進一步的確認全長Rad21與其兩種裂解產物對乳腺上皮細胞株形態上的影響，當分別大量表現全長Rad21、N端與C端Rad21重組蛋白於人類乳腺上皮細胞株MCF-7中，以螢光免疫染色發現全長的Rad21蛋白大多座落於細胞核中，N端與C端裂解產物則分布於細胞核與細胞質中。除此之外，當以轉染方式表現C端Rad21蛋白於MCF-7細胞株中時，經由辨認活化細胞凋亡的執行蛋白caspase-3的抗體作螢光免疫染色，可偵測到表現C端Rad21的細胞亦同時表現活化的caspase-3蛋白，這顯示C端Rad21蛋白能誘導MCF-7進行計畫性細胞凋亡。為確認Rad21被蛋白酶截切與caspase-3間在發育乳腺中的相關性，以誘導細胞凋亡的藥物etoposide處理不朽化的乳腺上皮細胞株MCF-10A，以活化caspase-3的表現。經由西方吸漬法證實當MCF-10A被誘導發生細胞凋亡時，外源性表現的全長的Rad21可被截切產生一相似於C端Rad21裂解產物大小的片段。 綜合以上結果，全長Rad21在泌乳末期與離乳初期會被截切成N端與C端蛋白質產物，並從細胞核轉移至細胞質中。而C端Rad21蛋白即可能去誘導乳腺進行細胞凋亡，維持在不同發育階段乳腺細胞數量的穩定性。Rad21 is one of the major cohesion subunits that holds chromatids together and controls separation of sister chromatids during mitosis. It is considered a novel nuclear caspase target molecule and plays a role in repairing DNA when it is damaged. Rad21 is cleaved by caspase-3 to promote apoptosis when breast cancer tumor suppressor protein (BRCA1) is phosphorylated in response to cell cycle and DNA damage in vivo. Unlike most other organs, development of the mammary gland occurs predominantly after birth. Once the gland is established, cycles of proliferation, functional differentiation, and apoptosis of alveolar epithelium occur repeatedly with each pregnancy. Thus, the regulation of apoptosis and cell proliferation closely affect alveolar epithelium development into normal acini or transforming into tumors during acinar formation. However, the function of Rad21 is not studied in detail in terms of mammary gland development. This study was undertaken to investigate the role of Rad21 and its cleavage products in mammary epithelial cells. 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. Two rabbit polyclonal peptide antibodies were developed that specifically recognizes the N-terminal or C-terminal of Rad21. To examine the specificity of these antibodies, full length, N and C-terminal Rad21 were transiently expressed in NIH3T3. The antibodies specifically recognized recombinant full length, N and C-terminal Rad21 proteins, as was detected using immuno-fluorescent staining techniques. The Rad21 expression profile was demonstrate with western blotting technique that was use to analyze Rad21 and its cleavage products’ protein expression profile in different stages of mice mammary gland development. The results showed that the full length Rad21 was detected in each developmental stage of mice mammary gland, however, it decreased from lactation day 14 to weaning day 4. On the other hand, cleaved N and C-terminal Rad21 increased from lactation day 14 to weaning day 2. These data indicate that Rad21 may have a special role to play in mammary gland development. To investigate the localization of Rad21 and its cleavage products in mammary epithelial cells, we transiently transfected full length N and C-terminal Rad21 into human mammary epithelial cell line MCF-7 and observed under immuno-fluorescent staining. The result suggested that the full length Rad21 was express only in the nucleus while N and C-terminal Rad21 were also present in the cytoplasm.Since the N and C-terminal Rad21 is highly expressed in late lactation and early weaning, we assume that they are involved in mammary gland involution. To address that, we transiently expressed Rad21 in MCF-7 cells and interestingly the data showed that only C-terminal Rad21 results in the induction of MCF-7 apoptosis as evidenced by the detection of active caspase-3 antibody with immuno-fluorescent staining. Furthermore, Rad21 was cleaved after inducing apoptosis by etoposide in immortalized human mammary epithelial cell line MCF-10A. Combined together, these results show that, full length Rad21 is cleaved to generate N and C-terminal Rad21 that again are translocated from nucleus to cytoplasm, with the C-terminal Rad21 involved in promoting apoptosis of mammary epithelial cells. These data hint that the expression of C-terminal Rad21 in weaning of mammary gland may have a role in the regulation of mammary gland development.目錄 頁次 壹、中文摘要 1 貳、英文摘要 3 參、緒言 6 肆、文獻探討 7 一、乳腺的發育 7 二、體外乳腺泡的形態發育與細胞凋亡調控 9 (一) 以體外3D培養模式及特殊蛋白標誌探討乳腺泡 的形成過程 9 (二) 細胞凋亡與乳腺泡形成之關係 11 三、乳癌的發生 11 (一) 非遺傳性乳癌 12 (1) 內泌素危險因子 12 (2) 非內泌素性危險因子 13 (二) 遺傳性乳癌與乳癌感受性基因BRCA1 14 四、乳癌與致癌基因之遺傳關係 15 五、增生性及抗細胞凋亡致癌基因與乳腺泡結構破壞之 關係 16 六、Rad21的功能 17 (一) Rad21為cohesin蛋白質複合體的一員 18 (二) Rad21的表現與損壞雙股DNA之修補 18 (三) Rad21的C端序列與細胞凋亡間之關係 19 (四) Rad21與癌症之相關研究 21 伍、材料與方法 23 一、乳腺組織total RNA之萃取 23 二、細胞total RNA萃取 23 三、反轉錄酶聚合鏈鎖反應 24 四、聚合酶鏈鎖反應 25 五、質體pGEM-T Easy-hRad21、hNRad21、hCRad21 的構築 27 六、細菌電穿孔轉型(electroporation)及X-gal誘發表現 藍白菌落 28 七、萃取小量質體DNA(DNA mini-extraction) 29 八、pCMV-3Tag hRad21、pCMV-3Tag hC-Rad21與 pCMV- 3Tag- hN-Rad21質體的構築 30 (1)限制酶截 30 (2)洋菜膠回收DNA¬¬ 30 九、中量質體DNA之萃取 31 十、細胞解凍 32 十一、細胞培養 32 十二、細胞繼代 32 十三、細胞轉染(Transfection) 33 十四、細胞免疫染色 34 十五、多株抗體製備 35 (1)抗原片段預測與合成 35 (2)Immunization 36 十六、細胞蛋白質萃取 36 十七、蛋白質定量 37 十八、西方吸漬法(Western blotting)與點墨吸漬法(Dot blotting) 37 陸、結果 40 一、抗原位置預測與兔子免疫反應 40 二、N端與C端Rad21抗體專一辨認其外源性蛋白質 41 三、N端與C端Rad21抗體專一辨認外源性全長Rad21蛋白質 43 四、Rad21差異表現於各時期乳腺組織 44 五、Rad21的C端裂解產物影響乳腺細胞骨架之分佈 46 六、Caspase-3截切全長Rad21產生C端Rad21於不朽化人類 乳腺上皮細胞株 47 七、C端Rad21誘導細胞凋亡於人類乳癌細胞株 48 柒、討論 50 捌、結論 55 玖、參考文獻 56 圖次 圖1. 人類、小鼠與牛間全長Rad21胺基酸序列保留性之比 較 60 圖2. 以DNA star軟體分析Rad21胺基酸序列預測可能抗原 片段 61 圖3. Rad21抗原施打與抗體力價檢測時間點流程圖 62 圖4. 利用點墨吸漬法對N端與C端Rad21抗體進行力價分 析 63 圖5. 抗Rad21 N端胜肽抗體辨認外源性N端Rad21蛋白質 能力之免疫螢光染色分析 64 圖6. 抗Rad21 C端胜肽抗體辨認外源性C端Rad21蛋白質 能力之免疫螢光染色分析 66 圖7. 抗Rad21 N端與C端胜肽抗體辨認外源性全長Rad21 蛋白質能力之免疫螢光染色分析 68 圖8. 抗Rad21 N端與C端胜肽抗體辨認外源性綠色螢光蛋 白能力之免疫螢光染色分析 70 圖9. Rad21蛋白質於不同發育時期小鼠乳腺中表現情形 71 圖10. N端Rad21蛋白質於不同老鼠乳腺時期表現情形 72 圖11. C端Rad21蛋白質於不同老鼠乳腺時期表現情形 73 圖12. 全長Rad21、N與C端Rad21蛋白質於不同成鼠乳腺發 育時期之表現情形 74 圖13. 全長、N端與C端Rad21蛋白對人類乳腺上皮細胞 MCF-7 actin的影響 76 圖14. Rad21的C端裂解蛋白對人類乳腺上皮細胞MCF-7 細胞骨架蛋白actin之影響 78 圖15. 全長、N端與C端Rad21蛋白對人類乳腺上皮細胞 MCF-7細胞骨架蛋白2303229 bytesapplication/pdfen-US乳腺姊妹染色分體乳癌細胞凋亡mammary glandRad21breast cancerapoptosis[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/56622/1/ntu-95-R93626014-1.pdf