摘要
ATM (Ataxia telangiectasia-muted)蛋白質在DNA damage repair、cell cycle arrest及細胞死亡的決定中扮演重要角色。它可藉由調控下游眾多基因而去完成不同的任務。然而，此蛋白在惡性腦瘤之低輻射敏感性所扮演之機制及角色目前仍不清楚。在基因治療之研究中，TFO (triplex forming oligonucleotide)及RNAi (RNA interference)兩個系統分別被認為具有在DNA層次及mRNA層次對特定目標基因作抑制之功能。然而,有關此兩系統的抑制效率到目前為止還沒有被比較過。因此，比較此兩種系統對於特定基因ATM之抑制，以及ATM基因在惡性腦瘤之低輻射敏感性中扮演的角色是非常值得研究的。為了研究TFO的功能，三組針對ATM基因特定序列之TFO分別被設計，並進行測試。首先，研究TFO之三股結合效率及溶點。結果顯示三組TFO皆具有與ATM目標基因之特定DNA雙股序列結合之能力，三股產物的溶點大於40℃。暗示了所設計之TFO系統在生物體中與特定目標基因形成穩定結合之可能性。接下來，在細胞實驗中觀測其傳送入細胞之傳輸效率及時程性抑制效率。在傳輸效率的實驗中顯示，TFO混合物加入細胞後12小時約90％以上的細胞質或核內都能觀測到TFO存在；特定基因之抑制效率實驗中，可發現送入TFO後24小時內對特定基因mRNA、 52小時內對特定基因蛋白質皆有顯著抑制效果。此外，三組TFO由於其組成不同，在時程性抑制效果中個別有不同的表現。相關研究中，這是首次使用TFO的系統來抑制ATM基因表現的實驗。在RNAi系統之建構方面，四組含有針對ATM基因特定序列shRNA之病毒載體分別被建構、純化及抑制效果測試。結果顯示四組shRNA序列片段在送入細胞12小時內對特定基因皆有顯著之抑制效果。挑選抑制效果最明顯之兩組RNAi及control序列，建構對應之長久穩定抑制型細胞株並分析特定基因表現量。結果顯示分別觀察不同代數之兩株細胞株特定基因之mRNA及蛋白質層次皆顯著及穩定地被抑制，相對的正常細胞及control RNAi細胞株則無被抑制現象。另外也觀察到，ATM穩定抑制型細胞株之生長速率與正常及control細胞相比明顯遲緩，細胞群落形態明顯不同，未受刺激之情況下細胞內之自由基含量明顯較高。最後，在這個研究裡探討了ATM之抑制對於照射輻射後之惡性腦瘤細胞U87MG存活之影響。存活率實驗結果顯示ATM之抑制能增加腦癌細胞U87MG之輻射敏感性，但對於人類子宮頸癌細胞Hela則較無顯著效果。另外，SLDR及PLDR之實驗結果顯示，ATM之抑制會影響此人類腦癌細胞SLDR及PLDR能力。以上存活率及SLD、PLD修復能力的實驗結果在相關研究當中是最新的發現。從以上的結果可以猜測ATM可能藉由SLDR及PLDR影響人類腦癌細胞U87MG之輻射敏感性，並可能與自由基之清除有關。然而，ATM基因在人類腦癌細胞之抗輻射特性中扮演之真實生物功能需要更近一步的實驗去證實。
並列摘要
ATM (Ataxia telangiectasia-muted) gene plays an important role in the control of cell cycle, signal transduction, DNA repair and cell death after cell damage by irradiation. The involving functions and the range are magnified by the downstreams of ATM gene. However, the mechanism and role of ATM in the radioresistance of brain cancer remained largely unclear. In studies of gene therapy, TFO (triplex forming oligonucleotide) system and RNAi (RNA interference) system are believed to have functions of gene inhibition in DNA and RNA level, respectively. However, there were no published data to compare their inhibition effects. This study aimed to compare the ATM inhibition by these two systems and investigate the role of ATM played in the response of glioma to radiation therapy. To study the function of TFO, three TFOs that target different sequences of ATM gene were designed and assayed. The results showed that three TFOs could bind to their duplex targets. The melting point of the triplex is greater than 37℃ which indicates the possibility of triplex formation within cells under normal physiological condition. Resuls also showed that TFO could be efficiently delivered into cells. Delivery efficiency assay indicated that TFO were found in almost 90％ cells 12 hr after TFO delivery. Gene inhibition time course analysis found that the maximum inhibition effects on mRNA and protein levels occurred at 24 hr and 48- 52 hr after TFO transfection, respectively. Besides, three TFOs had different time-course of inhibitions because of their different composition. Among relevant research, this is the first experiment using TFO system to inhibit ATM gene expression. Regarding the construction of RNAi system, four virus vectors containing shRNAs for ATM inhibition were constructed, purified, and assayed. All of four shRNA expression cassettes had significant inhibition effects on mRNA levels at 12 hr after transfection. Corresponding stable-inhibition cell lines were constructed and specifically inhibited ATM expression was determined. We have also found that ATM stable-inhibition cell line had a retarded growth and different colony morphology. Moreover, the ROS increased in cells with inhibited ATM. Finally, the survival of U87MG glioma with suppressed ATM after irradiation was determined. The lack of ATM expression led to increase radiosensitivity in glioma U87MG but not in cervical carcinoma Hela. Besides, ATM inhibition influenced SLDR and PLDR ability of U87MG. The above results suggested that ATM inhibition may enhance radiation killing on glioma cells (U87MG) by affecting its SLDR and PLDR ability, and be associated with antioxidant function. However, further in vivo experiments were required to better define the role of ATM in human brain cancer to radiation therapy.