神經膠質母細胞瘤（GBM）是由異常的星狀膠細胞所組成，在世界衛生組織中定義為第四級的膠質瘤，為最常見也是最惡性的原發性腦瘤，目前的研究顯示，得到神經膠質母細胞瘤的病人統計上中間的生存期( median survival )大約是15個月左右，5年以上的生存率小於4%。帝盟多（TMZ）是目前為止最為廣泛用於治療神膠質母細胞瘤的第一線用藥，目前的研究已表明帝盟多的治療可以誘導細胞凋亡，自體吞噬，細胞衰老和細胞週期阻滯。帝盟多的治療效果好壞很大的程度取決於O6-methylguanine–DNA methyltransferase (MGMT)這個蛋白質的表現量，不過目前對於神膠質母細胞瘤是如何抵抗帝盟多治療相關的機制還不太清楚。所以本實驗想要探討神經膠質母細胞瘤抵抗帝盟多治療的相關機制。我們使用1306-MG (人類神經膠質母細胞瘤細胞株)和CNS-1 (大鼠神經膠質母細胞瘤細胞株)這兩種不同的細胞株，來連續給予帝盟多並且利用細胞計數的方法篩選生長抑制50%存活下來的細胞而選出具有抗藥性的神膠質母細胞瘤細胞株。在建立具有帝盟多抗性的神膠質母細胞瘤細胞株的過程中，我們利用西方墨點法觀察MGMT的蛋白質表現量; 原位末端轉移酶技術和西方墨點法來觀察細胞凋亡; 利用免疫螢光染色和西方墨點法觀察細胞自噬作用; β-半乳糖苷酶活性比色法和西方墨點法觀察細胞老化; 細胞流式儀觀察細胞週期的改變; 明膠酶譜法觀察MMP-9的活性。在神膠質母細胞瘤細胞株對於帝盟多產生抗藥性的過程中我們發現MGMT蛋白質表現量在兩種細胞株中都沒有發生改變，細胞凋亡的比例降低，細胞老化的比例降低，細胞自蝕的比例增加，細胞週期的停滯從G2/M 時期進入G0/G1時期，MMP-9的活性增加，這也代表著MGMT在我們選用的細胞株中這個蛋白質未參與抗藥性產生的調控，而以上所觀察的現象改變可能參與抗藥性的產生。綜合以上之結果，可以發現細胞凋亡，細胞凋亡，細胞自蝕，細胞週期的停滯等改變都可能是導致神膠質母細胞瘤對於帝盟多產生抗藥性的重要原因。

WHO defines the Glioblastoma multiforme (GBM) as a grade IV glioma, which is the most common and hostile malignant primary brain tumor in human population. Previous studies showed that the GBM median survival is less than 15 months. The 5 year survival rate is less than 4%. Temozolomide (TMZ) is one of the most widely used drugs to treat GBM. Recently, it has been showed that TMZ treatment can induce apoptosis, autophagy, cell senescence and cell cycle arrest depend on MGMT expression level. However, the TMZ-induced resistance formation mechanism remains unclear. This study intends to establish two TMZ-induced resistance cell lines and test the role of apoptosis, cell senescence, autophagy and cell cycle arrest in their survival and death. The 1306-MG (human GBM), CNS-1 (rat GBM), and human astrocyte cell lines were used for establish TMZ-induced resistance (TIR) model. All of these cell lines were checked for MGMT expression level by immunoblotting. Trypan Blue Exclusion Assay (TBEA) method and cell counting were used to assess cell viability. Furthermore, TUNEL was used to observe apoptosis, LC3 staining was used to observe autophagy, SA-βGal was used to observe cell senescence, and zymography was used to observe MMP-9 activity, and cell flow-cytometry was used to determine cell cycle change. The MGMT expression levels of TIR GBM were not changed through generation of resistance. The cell viability increased through establishment of TIR accumulation under same concentrations of TMZ treatment; TIR significantly reduced the numbers of senescence cells in 1306-MG and CNS-1 TIR cell; the autophagy level and MMP-9 activity of TIR-GBM were elevated in 1306-MG and CNS-1 cells with generation of TIR cell; TIR cells showed significantly decreased SubG1 phase of cell cycle through generation of TIR cell, while both 1306-MG and CNS-1 showed decreased cell cycle distribution of S and G2/M phases. We had established 1306-MG and CNS-1 TMZ-induced resistance model. We also found that TIR formation accompanied with decreased apoptosis, diminished cellular senescence, arising autophagy, cell cycle arrest at G0/G1 phase, and increased MMP-9 activity. We conclude apoptosis, autophagy, cellular senescence, and cell cycle arrest all participate in the development of TMZ-induced resistance.

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