臺灣博碩士論文加值系統

在腦瘤的種類中，神經膠母細胞瘤是發生頻率最高的惡性原位腦瘤，而神經膠母細胞瘤的治療方式是以放射線治療和手術治療為主，但是近年來有放射線抗性的病人有增加的趨勢，科學家認為和癌症幹細胞有關。因此在癌症治療方面有很多學者在尋找可以抑制癌症幹細胞的藥物，藉此來增加病人對於放射線療法的敏感性。而靈芝被認為是靈丹妙藥而且使用了很多個世紀，而在過去有研究發現，靈芝多醣具有免疫調節和抑制腫瘤生長的效果。因此我們想要研究當靈芝多醣使用在神經膠母細胞瘤時，是否會有抑制神經膠母細胞瘤的效果出現。在我們的結果中有發現，當單獨使用靈芝多醣在神經膠母細胞瘤時，其MTT、cell cycle還有細胞死亡方面並沒有明顯的效果出現。因此，我們利用合併使用靈芝多醣和放射線治療，看是否對於神經膠母細胞瘤會有比較好的效果產生。在結果中可發現合併使用後，它會藉由抑制癌症幹細胞來達到腫瘤細胞減少。除此之外，我們也研究腫瘤細胞減少的原因，就可從結果知道合併治療可以透過MAPK和AKT路徑去導致細胞老化。因此，我們的結論是當在神經膠母細胞瘤上合併使用靈芝多醣和放射線時，其靈芝多醣會使神經膠母細胞瘤中的類癌症幹細胞特性減少後增加其放射敏感性，促使腫瘤細胞走向老化，使神經膠母細胞瘤細胞停止生長

The glioblastoma is the highest frequency of malignant primary brain tumor. In general, the main therapy of glioblastoma is radiation and surgery therapies. But more and more radioresistance patients are discovered in recent years. The researchers though that the radioresistance was related to cancer stem cell. So many papers investigated the cancer drugs which were the cancer stem cell inhibitor. These kinds of drugs could increase the radiosensitivity of patients. The herbal we used in this paper was Ganoderma lucidum. The Ganoderma lucidum was used in many centuries, and in previous studies found the extract of Reishi-derived polysaccharides (EORP) had immunomodulation and anti-tumor abilities. So we want to investigate the effect of EORP in glioblastoma. Our results revealed that when only treat EORP, the MTT assay、cell cycle and annexin-V stain all don’t have obvious significant results in gliobalsoma cell line. Therefore, we combined EORP and radiation to study the effect in radiation resistance glioblastoma cell line. The results show that the combination treatment can decrease the cell viability of glioblastoma cell, which by reduced stem-like cell property and markers in glioblastoma. It means that the EORP can affect the stem-like cell in glioblastoma and can increase the radiation sensitivity of glioblastoma. In addition, we further to investigate the reason for the decrease in cell viability. According to our results, we found that combination treatment could through MAPK and AKT pathway to induce cell senescence. In summary, our study shown that when combine with EORP and radiation can increase radiation sensitivity and induce cell senescence in glioblastoma cell line. It caused the glioblastoma cell viability to decrease in vitro.

中文摘要…………………………………………………………………………….. 1
Abstract……………………………………………………………………………… 2
Introduction…………………………………………………………………………. 4
Materials and Mwthods………………………………………………………..….. 11
Cell culture……………………………………………………...…………… 11
Antibodies………………………………………………………………......... 11
Preparation of EORP…………………………………………...………......... 11
Irradiation treatment…………………………………………………………. 12
MTT assay………………………………………………………………........ 12
Sphere assay……………………………………………………………......... 12
Cell cycle analysis…………………………………………………………… 13
Apoptosis analysis…………………………………………………………… 13
Preparation of whole cell extracts…………………………………………… 14
Western blotting analysis……………………………………………………. 14
SA-β-Gal staining…………………………………………………………… 15
Statistical analysis…………………………………………………………… 15
Result……………………………………………………………………………….. 16
Dose-dependent and time course effect of EORP on cell viability in S1R1,
human glioblastoma stem-like cell…………………………………………… 16
Dose-dependent and time course effect of EORP on cell cycle in S1R1…….. 16
Dose-dependent and time course effect of EORP on apoptosis of S1R1…….. 17
The combination of EORP with irradiation on S1R1 cell……………….…… 17
Dose-dependent effect of EORP on cancer stem-like cell properties
in S1R1……………………………………………………………………….. 18
Cell cycle distribution and cell death in S1R1 cells treated with
irradiation and EORP alone or in combination………………………………. 18
Senescence in S1R1 cells treated with irradiation and EORP alone
or in combination…………………………………………………………….. 19
The signaling molecules AKT and Erk involve in senescence in S1R1 cells
treated with irradiation and EORP alone or in combination…………………. 20
Discussion…………………………………………………………………………... 21
Reference…………………………………………………………………………… 26
Figure legend……………………………………………………………………….. 30
Figure 1. EOPR inhibit the S1R1 cell survival……………………………… 30
Figure 2. EORP can affect S1R1 cell cycle………………………………….. 31
Figure 3. EORP can’t affect S1R1 survival………………………………….. 34
Figure 4. EORP and irradiation can decrease S1R1 survival………………... 36
Figure 5. EORP affect the proteins for glioma stem –like cell………………. 37
Figure 6. Combination treatment can affect S1R1 cell cycle and survival…... 39
Figure 7. Combination treatment induce S1R1 senescence………………….. 42
Figure 8. Combination treatment affect S1R1 senescence pathway…………. 46

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