臺灣博碩士論文加值系統

放射治療對於子宮頸癌病人來說是具有影響性的治療之一，由其對於病人癌症細胞侵襲基底膜或是遠端轉移有不錯的療效。然而病人接受放射治療後所得到的癒後結果變化非常不一致。在本研究中，我們將探討子宮頸癌HeLa細胞在子宮頸癌HeLa細胞與基質細胞混和的培養液中對於輻射敏感性之影響。準備基質細胞培養液或是混合類型的細胞培養液(基質細胞: HeLa cells = 1:1)，基質細胞為來自於距離腫瘤近端(Sn)或是遠端基質細胞(Sf)。將子宮頸癌HeLa細胞培養在同類型或是混合類型的細胞培養液中四個小時之後再經過8Gy的放射線照射。我們發現不管是同類型(Sn或是Sf)或是混合類型(mixSn或是mixSf)的細胞培養液中均可以增加HeLa的增生及細胞存活的能力。
從即時定量聚合連鎖反應分析中，當HeLa細胞培養在同類型(Sn或是Sf)的細胞培養液中並照射8Gy放射線後，DNA修補基因-BTG2於基質細胞Sn或是Sf的細胞培養液中，在8小時及24小時有逐漸上升的現象。當HeLa細胞培養在混合類型(mixSn或是mixSf)的細胞培養液中照射8Gy放射線後，發現GADD45α於8小時及24小時有明顯下降的趨勢。
以上結果顯示距離腫瘤近端或是遠端基質細胞的培養液可以使HeLa細胞在照射8Gy放射線後可以幫助DNA受損修復的情形。將HeLa細胞培養在混合類型(mixSn或是mixSf)的細胞培養液中可以減少HeLa細胞的凋亡。
在蛋白質抗體晶片分析顯示，於距離腫瘤近端基質細胞及HeLa細胞混和距離腫瘤近端基質細胞的兩種培養液中發現 HGF, EGF, IGFBP-2 及 IGFBP-4。並且在HeLa細胞混和距離腫瘤近端基質細胞的培養液中IGFBP-3 及 PDGF-AA有表現量上升的情形。磷酸化的MAPKp38路徑於距離腫瘤近端基質細胞及HeLa細胞混和距離腫瘤近端基質細胞的兩種培養液中分別在兩小時及四小時表達量明顯上升。在控制組培養液(DMEM無血清)中磷酸化的MAPKp38路徑於四小時表達量上升，但是不明顯。
依據上述結果初步認為，蛋白質抗體晶片於在HeLa細胞混和距離腫瘤近端基質細胞的培養液中觀察到IGFBP-3及PDGF-AA，這或許可以調控磷酸化的MAPKp38後減少HeLa細胞的DNA受損以避免細胞死亡及增加細胞的增生及群落的能力。

Radiotherapy is one of effective treatments for patients with cervical cancers, especially for those with direct invasion or distal metastasis. However, outcome of radiotherapy is not consistent. In this study, role of stromal cells in radio-sensitivity of cervical cancer HeLa cells cultured in condition media (CMs) derived form coculture of HeLa cells and stromal cells were investigated. To prepare conditioned media from stromal cells or mixcultures (Stromal cells : HeLa cells = 1:1), stromal cells prepared from stromal cells closed to the cervical tumor were described as Sn, or those farther away from cervical tumor were described as Sf. HeLa cells were cultured in CMs derived from stromal cells or HeLa/stromal coculture for 4 hours then irradiated at 8Gy. Those CMs could increase proliferation and cell survival of HeLa cells after 8Gy irradiation.
Quantitative PCR (qPCR) revealed that expression of DNA damage repair gene, BTG2 was up-regulated gradually at 8hours and 24hours, and apoptosis gene, GADD45α was down-regulated at 8hours and 24hours after 8Gy radiation when HeLa cells were treated with CMs from mixSn and mixSf.
These results suggest that SnCM and SfCM may support DNA damage repair in HeLa cells irradiated with 8 Gy. MixSn and mixSf CMs may reduce apoptosis in HeLa cells with DNA damage. Protein antibody array indicated the presence of HGF, EGF, IGFBP-2 and IGFBP-4 in CMs derived from culture Sn and mixSn, and IGFBP-3 and PDGF-AA was up-regulated in mixSnCM. Phosphorylation of MAPKp38 (mitogen activated protein kinases) was upregulated at 2 and 4 hours in SnCM and mixSnCM.
In conclusion, cytokines such as IGFBP-3 in mixSnCM may regulate the phospho-p38 and decrease DNA damage in HeLa cells to avoid cell death after irradiation, therefore, increase proliferation and colony formation.

Content
致謝………………………………………………...………………….I
Abstract…………………………………………...……………….... II
中文摘要………………………………………………………….... IV
Introduction…………………………………………………………. 1
Materials…………………………………………………………….. 8
Methods…………………………………………………………..… 11
Results…………………………………………………………….... 18
Discussion……………………………………………………..….22
References…………………………………………………...……27
Appendix- Figure…………………………………………………...31
Appendix- Table………………………………………………….....37

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