臺灣博碩士論文加值系統

神經膠質母細胞瘤（GBM）是目前最常見且最惡性的原發性腦腫瘤。GBM難以治療的原因有很多，其中之一的原因為抗癌藥物難以遞送至癌細胞。在臨床上，抗癌藥物的投予通常在病患的腦水腫獲得改善之後。在本實驗室先前的研究中觀察到，GBM通常伴隨著腦水腫的症狀產生，多種發炎因子的表現量增加，以及血腦障壁的破損。而在臨床上治療GBM最常使用的藥物為帝盟多（Temozolomide, TMZ）. 帝盟多透過干擾癌細胞基因修復的能力去殺死癌細胞。在本實驗中，我們將實驗室先前建立的腦腫瘤引發腦水腫動物模型分成五組: (1)控制組；(2) 地塞米松處理組 (Dexamethasone, DEX)；(3) 帝盟多處理組；(4)臨床治療組；(5)共同治療組。先前的研究觀察到本動物模型在腫瘤細胞注射過七天後，腫瘤以及腦水腫仍然很小，但在第14天後急劇增長。所以本實驗將第2, 4, 5組的動物在腫瘤細胞處理過後第10到第12天使用腹腔注射，依序給予5mg, 2mg and 1mg/kg/day的地塞米松(DEX). 再將第3, 4, 5組的動物在腫瘤細胞處理過後第10到第16天使用腹腔注射，給予10mg/kg/day的帝盟多。僅有臨床治療組 (第四組) 在第13天到第16天時給予10mg/kg/day的帝盟多。接著使用核磁共振成像中的T2權重影像發現到控制組的腦腫瘤在第10天到第16天時快速增長，同時也觀察到腦水腫以及水腦症的病症以不同的程度出現在左右半腦。在動物醫學影像上的結果顯示，共同處理組的腦腫瘤跟腦水腫體積相較於控制組，地塞米松處理組，帝盟多處理組要來的小。並且共同處理組的動物平均生存天數皆優於其他組別。西方墨點法的結果顯示膠質原纖維酸蛋白(GFAP)的表現量再第13天跟第16天相互比較即可發現有經過治療的組別膠質原纖維酸蛋白的表現量有顯著性的趨緩(n=3, P〈0.01)。水通道蛋白-4(AQP4)的表現量在經過治療過後也有減緩的趨勢。腫瘤壞死因子-α以及介白素-1B等發炎因子的表現量更是在地塞米松以及共同處理組有顯著性大幅度的降低(n=3, P〈0.001)。在蘇木精-伊紅染色(H＆E staining)的結果顯示，在控制組腫瘤周邊有出血，鬆散的神經氈，以及腫瘤細胞浸潤等病理現象產生。而在共同處理組此些現象都有獲得相當良好的改善。透過免疫組織化學染色可以發現水通道蛋白-4跟膠質原纖維酸蛋白皆在腫瘤周邊大量表現，並有膠質細胞增生的病理特徵出現。而透過免疫螢光染色觀察鐵傳遞蛋白受體(Transferrin receptor, CD71)，可以觀察到使用地塞米松處理的組別血腦障壁破損的狀況有獲得改善。本研究提供了一些證據證實在由腫瘤引發的血腦障壁通透性增加期間同時投予地塞米松以及帝盟多可以達到最好的治療效果。共同治療組不僅僅在腦水腫以及腦腫瘤體積上跟其他組別相比有明顯性地降低，並且在動物生存天數上有顯著性的增長。在未來的治療腦腫瘤的選項上，可以考慮使用地塞米松以及帝盟多共同治療。

Glioblastoma multiforme (GBM) is the most common primary brain tumor. It is also the most aggressive and hard to be cured brain cancer. There are many reasons for unsatisfactory therapeutic outcomes in GBM. One of the main reasons is the difficulty to delivery drugs to the target cancer cells. In clinical setting, therapeutic agents are usually given to the patient after cerebral edema is under control. In the previous studies, we found cerebral edema often occurs along with GBM, and it causes blood-brain barrier (BBB) disruption and up-regulation of inflammatory cytokines. One of the most commonly used drugs in GBM is Temozolomide (TMZ), which kills cancer cell by interfering with tumor DNA repair. It is offen used in patients after the glioma induced edema is controlled with dexamethasone (DEX, a steroid). In this study, we hypotheses the efficacy of TMZ treatment may be enhanced if it is used in edema stage. To clarify our hypothesis, we use a GBM-induced cerebral edema animal model which established previously, and we divide the rats into five groups: (1) control group; (2) GBM-induced edema animal model treated with DEX; (3) GBM-induced edema animal model treated with TMZ; (4) GBM-induced edema animal model treated with DEX and TMZ separately to mimic clinic setting; (5) GBM-induced edema animal model co-treated with DEX and TMZ. Our previous studies showed that the sizes of brain tumor and cerebral edema were small on day 7 after tumor graft, but were well developed on 14 days after tumor injection. Therefore, in group 2, 4 and 5, animals were treated with DEX on day10 to day12 with the following dosages: 5mg, 2mg, and 1mg/kg/day. In group 3 and 5, animals were treated with TMZ on day10 to day16 with a dosage of 10mg/kg/day. Only the animals in group 4, the separated treatment group, were treated with TMZ on day13 to day16. The T2-weight MRI images showed that the tumor size and the cerebral edema area of control group increased rapidly on day 10 to day 16, and we found hydrocephalus which appeared at the right ventricle and extending to the left side. The images also showed the tumor size and cerebral edema area of group 5 were smaller than group 1, 2 and 3, which corresponded to the increased survival time of group 5. According to the results, we conclude that cancer-induced complication of hydrocephalus and cerebral edema may lead to death of the control group. Our results also showed that the efficacy of co-treatment with DEX and TMZ was better than DEX alone (group 2) and TEX alone (group 3). The results of western blot showed the expression of GFAP and AQP4 decreased significantly with treatments from day 13 to day 16 (n=3, P〈0.01). The expression of inflammatory cytokines, TNF-α and IL-1β, decreased significantly in DEX alone and co-treatment group (n=3, P〈0.001). Furthermore, the results of H＆E staining images revealed hemorrhages, loose neuropil around tumor to suggest edema, and tumor cell infiltration in control group. These pathology phenomenons were improved at co-treatment group. The IHC staining results showed that AQP4 and GFAP overexpressed around the tumor, and pathological gliosis appeared in the brain parenchyma all groups. The Immunofluorescence (IF) staining of CD71 showed evidences that BBB disruption at group 1, 3 and 4, but was improved after DEX treatment at group 2 and 5. This study provides evidences that co-treatment with DEX and TMZ presents with significantly improved therapeutic effect. The co-treatment group showed significantly decreased in edema and tumor size when compared with other groups. The average survival day of the co-treatment group was significantly longer than that of the other groups. Therefore, co-treatment of DEX and TMZ may consider as a therapeutic option for treating GBM in the future.

Table of Contents
Abstract in Chinese.........III
Abstract in English.........V
Acknowledgment.........VII
Table of Contents.........IX
Introduction..........1
Glioblastoma Multifirme (GBM).........1
Brain Edema...........1
Blood-Brain Barrier..........2
Hydrocephalus..........2
Aquaporin 4..........3
Dexamethasone..........3
Temozolomide...........4
Brain Inflammatory Cytokine........5
Clinical Treatment of Glioblastoma Multiforme.....6
Aim And Experimental design of the Study.......7
Experimental Design.........8
Time Couse Experiment.........9
Materials and Methods..........10
Cell Culture.........10
Established GBM-induced Brain Edema Model.....10
Temozolomide (TMZ) and Dexamethasone (DEX).....11
Tissue Processing.........11
H＆E Staining.........12
Immunohistochemical Staining......12
Double Immunofluorescence Staining......12
Protein Purification........13
Western Blot..........13
T2-weight Magnetic Resonance Imaging.....13
Statistical analysis.........14
Results...........15
The MRI T2-weight images after tumor injection......15
The development of tumor were edema mass.......15
The expression of AQP4, GFAP, TNF-α, IL-1β and IFN-γ...16
Brain Tumor Morphology........17
The IHC staining of AQP4 and GFAP.......18
The Disruption of Blood-Brain Barrier........18
Discussion..........20
Tables..........23
Figures...........25
Reference...........43

References
1.Johnson DR, O’Neill BP. Glioblastoma survival in the United States before and during the temozolomide era. J Neurooncol. 107(2):359-64 (2012)
2.Fukuda AM, Adami A, Pop V, et al. Posttraumatic reduction of edema with aquaporin-4 RNA interference improves acute and chronic functional recovery. J Cereb Blood Flow Metab. 33(10):1621-32 (2013)
3.Friedman B, Schachtrup C, Tsai PS, et al. Acute vascular disruption and Aquaporin 4 loss after stroke. Stroke. 40(6):2182-2190 (2009)
4.Ropper AH, Shafran B. Brain Edema After Stroke. Arch Neurol. 41(1):26-29 (1984)
5.Tang Y, Wu P, Sua J, et al. Effects of Aquaporin-4 on edema formation following intracerebral hemorrhage. Exp Neurol. 223(2):485-95 (2010)
6.Groothuis DR. The blood-brain and blood-tumor barriers: a review of strategies for increasing drug delivery. Neuro Oncol. 2(1):45-59 (2000)
7.Belayev L, Busto R, Zhao W, et al. Quantitative evaluation of blood-brain barrier permeability following middle cerebral artery occlusion in rats. Brain Res. 739(1-2):88-96 (1996)
8.Melo GD, Grano FG, Silva JE, et al. Blood-brain barrier disruption during spontaneous canine visceral leishmaniasis. Parasite Immunol. 37(12):635-45 (2015)
9.Del Bigio MR. Neuropathological changes caused by hydrocephalus. Acta Neuropathol. 85(6):573-85 (1993)
10.Papadopoulos MC, Verkman AS. Aquaporin-4 and brain edema. Pediatr Nephrol. 22(6):778-84 (2007)
11.Papadopoulos MC, Manley GT, Krishna S, et al. Aquaporin-4 facilitates reabsorption of excess fluid in vasogenic brain edema. FASEB J. 18(11):1291-3 (2004)
12.Yang L, Wang X, Zhen S, et al. Aquaporin-4 upregulated expression in glioma tissue is a reaction to glioma-associated edema induced by vascular endothelial growth factor. Oncol Rep. 28(5):1633-8 (2012)
13.Fukuda AM, Adami A, et al. Posttraumatic reduction of edema with aquaporin-4 RNA interference improves acute and chronic functional recovery. J Cereb Blood Flow Metab. 2013 Oct;33(10):1621-32.
14.Tang Y, Wu P, Sua J, et al. Effects of Aquaporin-4 on edema formation following intracerebral hemorrhage. Exp Neurol. 223(2):485-95 (2010)
15.Gobiet W, Bock WJ, Liesegang J, et al. Treatment of acute cerebral edema with high dose of dexamethasone. Intracranial Pressure III. 231-235 (1976)
16.Fan Z, Sehm T, Rauh M, et al. Dexamethasone alleviates tumor-associated brain damage and angiogenesis. PLoS One. 9(4):e93264 (2014)
17.Gamache DA. Ellis EF. Effect of dexamethasone, indomethacin, ibuprofen, and probenecid on carrageenan-induced brain inflammation. J Neurosurg. 65(5):686-92 (1986)
18.Sáez-Llorens X, Jafari HS, Severien C, et al. Enhanced attenuation of meningeal inflammation and brain edema by concomitant administration of anti-CD18 monoclonal antibodies and dexamethasone in experimental Haemophilus meningitis. J Clin Invest. 88(6): 2003-2011 (1991)
19.Ohnishi T, Sher PB, Posner JB, et al. Capillary permeability factor secreted by malignant brain tumor. Role in peritumoral brain edema and possible mechanism for anti-edema effect of glucocorticoids. J Neurosurg. 72(2):245-51 (1990)
20.Heiss JD, Papavassiliou E, Merrill MJ, et al. Mechanism of dexamethasone suppression of brain tumor-associated vascular permeability in rats. Involvement of the glucocorticoid receptor and vascular permeability factor. J Clin Invest. 98(6):1400-8 (1996)
21.Arya SK, Wong-Staal F, Gallo RC. Dexamethasone-mediated inhibition of human T cell growth factor and gamma-interferon messenger RNA. J Immunol. 133(1):273-6 (1984)
22.Larsson EL. Cyclosporin A and dexamethasone suppress T cell responses by selectively acting at distinct sites of the triggering process. J Immunol. 124(6):2828-33 (1980)
23.Auphan N, DiDonato JA, Rosette C, et al. Immunosuppression by glucocorticoids: inhibition of NF-kappa B activity through induction of I kappa B synthesis. Science 270(5234):286-90 (1995)
24.Hughes MA, Parisi M, Grossman S, et al. Primary brain tumors treated with steroids and radiotherapy: low CD4 counts and risk of infection. Int J Radiat Oncol Biol Phys 62(5):1423-6 (2005)
25.Grossman SA, Ye X, Lesser G, et al. Immunosuppression in patients with high-grade gliomas treated with radiation and temozolomide. Clin Cancer Res 17(16):5473-80. (2011)
26.Wong ET, Lok E, Gautam S, et al. Dexamethasone exerts profound immunologic interference on treatment efficacy for recurrent glioblastoma. Br J Cancer 113(2):232-41 (2015)
27.Denny BJ, Wheelhouse RT, Stevens MF, et al. NMR and molecular modeling investigation of the mechanismof activation of the antitumor drug temozolomide and its interaction with DNA. Biochemistry 33(31):9045-9051 (1994)
28.Tisdale MJ. Antitumor imidazotetrazines--XV. Role of guanine O6 alkylation in the mechanism of cytotoxicity of imidazotetrazinones. Biochem. Pharmacol. 36(4): 457-462 (1987)
29.Groot JF, Wen PY, Lamborn K, et al. Phase II single arm trial of aflibercept in patients with recurrent temozolomide-resistant glioblastoma: NABTC 0601. Journal of Clinical Oncology, 2008 ASCO Annual Meeting Proceedings (Post-Meeting Edition).Vol 26, No 15S (May 20 Supplement), 2008: 2020 (2008)
30.Monti D, Troiano L, Tropea F, et al. Apoptosis--programmed cell death: a role in the aging process? Am J Clin Nutr. 55(6 Suppl):1208S-1214S (1992)
31.Knizhnik AV, Roos WP, Nikolova T, et al. Survival and death strategies in glioma cells: autophagy, senescence and apoptosis triggered by a single type of temozolomide-induced DNA damage. PLoS One 8(1): e55665 (2013)
32.Ziegler DS, Kung AL, Kieran MW. Anti-apoptosis mechanisms in malignant gliomas. J Clin Oncol. 26(3):493-500 (2008)
33.Spiegl-Kreinecker S, Buchroithner J, Elbling L, et al. Expression and functional activity of the ABC-transporter proteins P-glycoprotein and multidrug-resistance protein 1 in human brain tumor cells and astrocytes. J Neurooncol. 57(1):27-36 (2002)
34.Jacinto FV, Esteller M. MGMT hypermethylation: A prognostic foe, a predictive friend. DNA Repair. 6(8):1155-60 (2007)
35.Wei KC, Chu PC, Wang HY, et al. Focused ultrasound-induced blood-brain barrier opening to enhance temozolomide delivery for glioblastoma treatment: a preclinical study. PLoS One. 8(3):e58995 (2013)
36.Vitkovic L, Bockaert J, Jacque C. Inflammatory cytokines: neuromodulators in normal brain? J Neurochem. 74(2):457-71 (2000)
37.Willenborg DO, Fordham S, Bernard CC, et al. IFN-gamma plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. J Immunol. 157(8):3223-7 (1996)
38.Chung IY, Benveniste EN. Tumor necrosis factor-alpha production by astrocytes. Induction by lipopolysaccharide, IFN-gamma, and IL-1 beta. J Immunol. 144(8):2999-3007 (1990)
39.Schindler R, Mancilla J, Endres S, et al. Correlations and interactions in the production of interleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood. 75(1):40-7 (1990)
40.Feuerstein GZ, Liu T, Barone FC. Cytokines, inflammation, and brain injury: role of tumor necrosis factor-alpha. Cerebrovasc Brain Metab Rev. 6(4):341-60 (1994)
41.Gery I, Gershon RK, Waksman BH. Potentiation of the T-lymphocyte response to mitogens. I. The responding cell. J Exp Med. 136(1):143-155 (1972)
42.Gray PW, Goeddel DV. Structure of the human immune interferon gene. Nature 298(5877):859-63 (1982)
43.Créange A, Barlovatz-Meimon G, Gherardi RK. Cytokines and peripheral nerve disorders. Eur Cytokine Netw. 8(2):145-51 (1997)
44.Curzio Rüegg, Aysim Yilmaz, Grégory Bieler, et al. Evidence for the involvement of endotheliai cell integrin αVβ3 in the disruption of the tumor vascuiature induced by TNF and IFN-γ Nature Medicine 4:408-414 (1998)
45.Stover JF, Schöning B, Beyer TF, et al. Temporal profile of cerebrospinal fluid glutamate, interleukin-6, and tumor necrosis factor-alpha in relation to brain edema and contusion following controlled cortical impact injury in rats. Neurosci Lett. 288(1):25-8 (2000)
46.Silbergeld DL, Rostomily RC, Alvord EC Jr. The cause of death in patients with glioblastoma is multifactorial: clinical factors and autopsy findings in 117 cases of supratentorial glioblastoma in adults. J Neurooncol. 10(2):179-85 (1991)
47.Bruce JN, Kenned BJ. Brain vancer treatment protocols. Medspace. Sep 12, 2013, from http://emedicine.medscape.com/article/2005182-overview
48.Bruce JN. Glioblastoma multiforme treatment ＆ management. Medspace. Aug 13, 2015, from http://emedicine.medscape.com/article/283252-treatment
49.Paul C, Bolton C. Inhibition of blood-brain barrier disruption in experimental allergic encephalomyelitis by short-term therapy with dexamethasone or cyclosporin A. Int J Immunopharmacol. Jun;17(6):497-503(1995)
50.Roger S, Monika EH, Warren PM, et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. The LANCET Oncology. Volume 10, No. 5, p459–466(2009)
51.Fertsch D, Schoenberg DR, Germain RN, et al. Induction of macrophage Ia antigen expression by rIFN-gamma and down-regulation by IFN-alpha/beta and dexamethasone are mediated by changes in steady-state levels of Ia mRNA. The Journal of Immunology , vol. 139 no. 1 244-249 (1987)
52.Sadhak S, Jaclyn M, Caroline F, et al. Impact of temozolomide on immune response during malignant glioma chemotherapy. Clinical and Developmental Immunology Volume 2012 (2012)
53.Zhang HH, Kumar S, Barnett AH, et al. Dexamethasone inhibits tumor necrosis factor-alpha-induced apoptosis and interleukin-1 beta release in human subcutaneous adipocytes and preadipocytes. J Clin Endocrinol Metab. Jun;86(6):2817-25(2001)
54.Monica AL, Danielle LI, Mariano TM, et al. Effect of dexamethasone administered with magnesium sulfate on inflammation-mediated degradation of the blood–brain barrier using an in vitro model. April; 21(4): 483–491(2014)