臺灣博碩士論文加值系統

研究證實癌細胞可透過釋放免疫抑制型激素，如TGF-β (transforming growth factor-β)，抑制自然殺手細胞 (natural killer cell, NK cell) 毒殺功能，進而躲避宿主免疫監控。TGF-β的大量表現於乳癌、大腸癌、胃癌等疾病的進程、轉移、不良預後皆具高度相關性。近年來的研究更證實在慢性骨髓性白血病中，TGF-β會保有白血病幹細胞之幹性 (stemness) 以維持其癌化能力 (tumorigenic activity)。目前科學家們正積極發展以TGF-β為目標的標靶療法，期望能將之運用於臨床治療上。另一方面，於先天性免疫反應中，NK細胞針對病毒感染和腫瘤細胞的清除有著舉足輕重的地位，其透過細胞表面的受體與目標細胞上之配體結合來執行免疫監控。與其他研究結果相似，我們的實驗亦發現經TGF-β作用後，NK細胞表面上的活化型受體會逐漸減少，且其對於白血病細胞之毒殺能力則大幅降低，指出白血病患者之免疫系統會受TGF-β影響而無法有效進行免疫監控之功能。此外，為了逃脫NK細胞的毒殺，研究證實急性骨髓性白血病患者的白血球芽細胞會藉由改變細胞表面相對應配體的表現量來抑制NK細胞之毒殺作用。基於上述研究發現，我們欲檢測TGF-β於白血病細胞對NK細胞毒殺能力感受性之影響，以驗證TGF-β的表現與白血病細胞躲避NK細胞的免疫監控之相關性。實驗結果發現，經TGF-β處理後，白血病細胞對NK細胞之毒殺感受性大幅降低。此外，TGF-β亦影響白血病細胞表面之醣化作用及其相對應NK活化型受體之配體的表現量，其中以CD48表現量的下降最為顯著。已知CD48可與NK細胞表面上的活化型受體2B4結合，啟動下游的訊息傳遞路徑。故在抑制CD48表現的細胞中亦觀察到NK細胞與之結合能力有顯著的下降，推測白血病細胞可能透過釋放TGF-β調控CD48-2B4訊息傳遞路徑以達到逃脫NK細胞毒殺作用之目的。我們期望能夠透過此研究提供另一訊息傳遞機制作為標靶治療開發之選擇。

Transforming growth factor-β (TGF-β)-mediated signaling has been reported in the regulation of tumor initiation, progression and metastasis in solid tumor. It has been known for decades that TGF-β attracts immune components into the tumor microenvironment to enable the expression of additional tumor-promoting factors. Furthermore, TGF-β has been proved the tumorigenic activity of leukemia-initiating cells in chronic myeloid leukemia (CML). Based on these findings, it would be a great idea to develop TGF-β-associated cancer therapy. On the other side, natural killer cells (NK cells), member of innate immune system, are critical to destroy virus-infected cells and tumor cells. NK cells processed immunesurveillance through recognizing ligand of target cells. With the agreement to other studies, our data showed that the cytotoxicity was impaired upon TGF-β treatment, which indicated that the function of immune cells were suppressed in the tumor microenvironment. Another research demonstrated the escaping NK-mediated cytotoxicity due to the surface ligand changing in acute myeloid leukemia (AML) patient. In combination, our hypothesis is that the TGF-β plays positive role on escaping NK-mediated surveillance of the leukemia cells. Our results showed that the susceptibility of TGF-β-treated leukemia cells to NK-mediated cytotoxicity decreased and the expression of tumor-associated carbohydrate antigens and the amount of corresponding ligands for NK activating receptors also been affected, especially on the expression of CD48. Furthermore, the established CD48-knockdown cells with decreasing levels of CD48 possessed lower NK sensitivity and less conjugation with NK-92MI cells, suggested the TGF-β-mediated signaling affects the susceptibility of leukemia cells through CD48-2B4 signaling pathway to help the escape from the NK-mediated immunosurveillance. Based on these findings, targeting TGF-β signaling may serve as another choice for developing the target therapy.

中文摘要.....i.
Abstract.....ii.
縮寫表.....iv.
目錄.....vi.
第一章 緒論.....1
1. 轉化生長因子 -β ( transforming growth factor-beta, TGF beta).....1
2. 白血病的成因與治療.....2
3. CD48.....4
4. 自然殺手細胞 (natural killer cells, NK cells).....5
第二章 研究動機與目的.....8
第三章 材料和方法.....9
1. 細胞培養.....9
2. 細胞接合力試驗 (conjugate formation assay).....9
3. NK 細胞去顆粒作用試驗 (NK degranulation assay).....10
4. 細胞毒殺試驗(FACS -based cytotoxicity).....10
5. 白血病細胞表面醣抗原和自然殺手受體相對應配體之染色.....11
6. NK 細胞表面受體之染色.....11
7. 非放射線細胞毒殺試驗.....12
8. 即時定量 PCR (real -time time qPCR).....12
9. 慢病毒表現系統 (lentivirus expression system).....13
10. 統計分析.....13
第四章 結果.....14
1. TGF-β 降低自然殺手細胞對目標細胞之毒殺能力.....14
2. TGF-β影響自然殺手細胞表面活化型受體之表現量.....14
3. TGF-β影響人類白血病細胞株對於 NK 細胞毒殺之感受性.....15
4. TGF-β 影響白血病細胞株表面醣抗原的表現量.....16
5. TGF-β 降低白血病細胞上 NK 活化型受體之相對應配體的表現量.....16
6. 抑制 CD48表現量之細胞對 NK 細胞之毒殺感受性顯著下降.....17
第五章 討論.....19
第六章 圖.....22
圖 1 TGF-β 影響 NK -92MI細胞之功能.....22
圖 2 TGF-β 影響 NK 細胞表面上之受體表現量.....24
圖 3 TGF-β 影響血癌細胞對於 NK-92MI 細胞之毒殺感受性.....26
圖 4 TGF-β 影響人類白血病細胞株表面上醣抗原和 NK 活化型受體之 相對應配體之表現.....29
圖 5 抑制 CD48 表現量影響人類白血病細胞株對 NK 細胞之毒殺感受性...31
參考文獻.....33

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