臺灣博碩士論文加值系統

KIAA1618/ALO17是在異生性大細胞淋巴瘤 (Anaplastic large cell lymphoma, ALCL) 病例中所發現的功能未知蛋白，由1063個胺基酸組成，在這些ALCL病例中，KIAA1618的N端 (胺基酸序列1-1008) 與異生性淋巴瘤激酶 (Anaplastic lymphoma kinase, ALK) 的C端 (胺基酸序列 1058-1620 ) 形成新的融合蛋白，根據先前的研究，融合蛋白與致癌性有密切的關係。為了研究KIAA1618的功能，將含有HA-tag KIAA1618的質體DNA轉染HEK293T細胞，利用免疫沉澱收取表現蛋白，根據銀離子染色與西方墨點法的結果，KIAA1618表現在接近170 kDa的位置，比預測大小118.43 kDa來的高。分層萃取與免疫螢光染色顯示KIAA1618主要分布於細胞質。利用In vivo ubiquitination assay，顯示在MG132 (proteasome inhibitor) 處理下，KIAA1618具有多泛素化的特性，而利用誘發內質網壓力的藥物也可以發現類似的特性。進一步，凋亡細胞偵測顯示大量表現KIAA1618的HeLa細胞對於Tunicamycin (N-glycosylation inhibitor)與Thapsigargin (Ca2+ pump inhibitor) 處理有較高的耐受性；比較在兩種藥物處理下蛋白質的表現量，結果發現在大量表現KIAA1618的HeLa細胞，Grp78的表現量和PARP-1的斷裂都有顯著降低。總結實驗結果，KIAA1618可能會增加細胞對內質網壓力的耐受性。

KIAA1618/ALO17 is a novel protein discovered from the cases of the anaplastic large cell lymphoma (ALCL). A chimeric protein created due to a translocation of the N-terminal KIAA1618 protein (a.a. 1-1008) fused with the C-terminal truncated anaplastic lymphoma kinase (ALK, a.a. 1058-1620). According to the reported cases of ALCL, the oncogenesis is highly correlated with this chimeric protein. Nonetheless, the physiological function of KIAA1618 is still remained unclear. Using HA-tagged KIAA1618 to transfect HEK293 cells, silver-stain and Western blotting results showed that KIAA1618 migrated at approximate 170 kDa on SDS-PAGE which is much higher than an estimated 118.43 kDa from the predicted 1063 amino acids of KIAA1618 cDNA. Subcellular fraction and immunofluorescence staining revealed a predominant cytosolic distribution of KIAA1618. In vivo ubiquitination assay showed that KIAA1618 was poly-ubiquitinated with the addition of MG132, a proteasome inhibitor. A similar result was found when cells were exposed to some ER stress drugs. Moreover, apoptotic detections showed that KIAA1618 overexpressing HeLa cells were resistant to Tunicamycin and Thapsigargin treatment, an N-glycosylation inhibitor and a Ca2+ pump inhibitor, compared with control cells. Finally, a significant decrease in the expression level of Grp78 and PARP-1 cleavage was found in KIAA1618 overexpressing compared with control cells treated with Tunicamycin and Thapsigargin. Collectively, these results suggested that KIAA1618 may render cells resistance to ER stress.

中文摘要………………………………………………………………………….........i
英文摘要……………………………………………...……………………………….ii
縮寫表………………………………………………………………………………...iii
目錄………………………………………………………………………………….. .v
第一章 前言…………………………………………………………………………..1
1.1 異生性大細胞淋巴瘤(Anaplastic large cell lymphoma，ALCL)…………1
1.2 異生性淋巴瘤激酶 (Anaplastic lymphoma kinase，ALK)……………….2
1.3 ALK融合蛋白………………………………………………………………3
1.4 ALK融合蛋白的致癌性訊息傳遞…………………………………………4
1.5 KIAA1618/ALO17 (ALK lymphoma oligomerization partner on chromosome 17)…………………………………………………………………6
1.6 研究動機……………………………………………………………............7
第二章 材料與方法…………………………………………………………………..8
2.1.1 細胞株培養……………………………………………………………….8
2.1.2細胞計數…………………………………………………………………..8
2.1.3 加藥處理………………………………………………………………….8
2.2.1 細胞蛋白質萃取 (Whole cell extract)………………………………...…9
2.2.2 染色質分離 (Chromatin fractionation)…………………………………..9
2.3.1 凋亡細胞偵測：Propidium Iodide核酸染色..........................................10
2.3.2 凋亡細胞偵測：FITC-Annexin V/Propidium Iodide雙重染色................10
2.4 免疫沉澱…………………………………………………………..……..11
2.5 免疫螢光染色……………………………………………………..……..12
2.6.1 基因轉染………………………………………………………………...12
2.6.2 大量表現細胞株建立…………………………………………………...13
2.6.3 In vivo ubiquitination assay……………………………………………...13
2.7.1 SDS-PAGE膠體電泳……………………………………………………14
2.7.2 西方墨點法…………………………………………………………...…15
2.7.3 銀離子染色…………………………………………………………...…15
2.8 In gel digestion…………………...……………………………………..16
第三章 實驗結果……………………………………………………………………17
3.1 KIAA1618蛋白質的表現大小……………………………………………17
3.2 KIAA1618在細胞內的分布………………………………………………17
3.3 透過LC-MS/MS尋找KIAA1618可能的關連蛋白…………………….18
3.4 KIAA1618具有泛素化的特性…………………….....................………18
3.5 大量表現KIAA1618的HeLa細胞受到Tunicamycin與Thapsigargin處理時的反應…................................................................................................….19
3.6 大量表現KIAA1618的HeLa細胞受到Tunicamycin與Thapsigargin處理時的蛋白表現量………………....................................…………...………..20
第四章 總結與討論…………………………………………………………………22
第五章 實驗結果圖表………………………………………………………………25
附錄…………………………………………………………………………………..37
參考文獻……………………………………………………………………………..40

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