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研究生:林彥仰
研究生(外文):Yen-Yang Lin
論文名稱:研發川芎嗪的新應用探討川芎嗪在神經膠細胞瘤的療效
論文名稱(外文):The New Application of Tetramethylpyrazine to Therapy of Glioma
指導教授:傅毓秀傅毓秀引用關係
指導教授(外文):Yu-Show Fu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:解剖暨細胞生物學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:77
中文關鍵詞:川芎神經膠細胞瘤
外文關鍵詞:gliomaTMP
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根據世界衛生組織(WHO) 宣布的腦瘤種類有9種,其中由腦細胞衍生而來的腫瘤,基本上可分為Neuroma 和Glioma 兩類。Glioma 是最常見的種類,Glioma 早期不容易診斷出來,並且生長速度很快,即使手術後效果也非常差,因此直到目前為止,Glioma 在臨床醫學上,屬於相當棘手也莫可奈何的神經疾病。Glioma 可能由正常之神經膠細胞病變而來,可發生在不同腦區,隨著腫瘤逐漸長大,便會造成腦壓升高、頭痛、嘔吐、失明、癲癇等異常行為,其存活期不超過一年(Benedetti et a.,2000)。
先前的研究指出,Glioma cells會釋出大量的Glutamate,導致周邊Glutamate濃度大幅上升,因此造成鄰近的神經細胞興奮性毒性,神經細胞的受損更幫助glioma cells的生長速率(Takano et al., 2001)。首先,我們必需盡量減低神經細胞在遭受興奮性毒性時的損傷,如此應可以抑制glioma cells的生長。在另一篇研究指出,glioma細胞內Ca2+濃度的上升,會增加細胞的活性、進而幫助glioma cells的migration,因此他們給予Ca2+ channel blocker (MK801),則能有效抑制Glioma細胞的增殖速率(Ishiuchi et al., 2002)。然而MK801為NMDA receptor的antagonist,一旦給予病人服用,則副作用太大,因此我們需要找另一個藥物來抑制Ca2+流入glioma cells內。
TMP是中藥川芎的主要成分之一,臨床上常採用川芎來治療腦血管與心血管栓塞的疾病,相關研究證實TMP有抑制外鈣內流的作用(Pang et al., 1996)。有關TMP對神經細胞的作用,我們實驗室曾證實TMP藉由保護粒線體、抑制自由基生成,降低kainate所誘發的神經細胞興奮毒性(Shih et al., 2002)。並且TMP確實可以通過血腦障壁(Tsai and Liang, 2001)。
基於以上特性,我們推測TMP有治療腦瘤的潛力,因此本實驗主要目的是探討TMP對C6 glioma細胞的治療效果及作用機制。首先,我們以不同濃度TMP處理培養中的C6 glioma細胞,實驗結果顯示,400μM TMP可以有效抑制腫瘤細胞生長,200μM及400μM TMP可以造成30%腫瘤細胞呈現受損的情形,50μM TMP即可避免腫瘤細胞內的鈣離子濃度因glutamate刺激而增加,並且降低腫瘤細胞glutamate的釋放,因此有效降低培養液中glutamate濃度。進一步,將腫瘤細胞和神經細胞共同培養,由TUNEL染色發現,神經細胞受損比例因此提高,導致神經膠瘤細胞繁殖速率增加為140%左右;以低濃度TMP處理(12.5μM-100μM),即可保護神經細胞,避免遭受神經膠瘤細胞的興奮毒性傷害。接著,將106神經膠瘤細胞植入大白鼠額葉皮質內,結果顯示,TMP有效延長腫瘤大白鼠的存活天數,並且減低腫瘤細胞的生長和活性。綜合我們的實驗結果,TMP有效抑制腫瘤細胞的生長,提供一個治療腦瘤的新藥物。
According to the publications of the World Health Organism (WHO), there are nine types of brain tumors. Glioma is the most frequently found brain tumor, and cannot be easily diagnosed at its early stage, and it grows quickly. Although glioma can be removed through operation, the effect of operation is generally unsatisfactory. Up to the present, glioma is still considered clinically as a problematic neurodisease. Glioma may be resulted from mutation of normal glial cells in various brain regions. The growth of the tumor is accompanied with pathological symptoms such as elevated brain pressure, headache, vomit, loss of sight and epilepsy. Most patients die within one years after the onset of glioma (Benedetti et al., 2000.
A previous study indicates that glioma cells release a large amount of glutamate, resulting in an elevated glutamate concentration in the periphery. The elevated glutamate concentration results in excitotoxicity to the surrounding neurons, and the damage to the neurons in turn promotes the growth of the glioma cells (Takano et al., 2001). Therefore, the reduction of the damage resulted from the excitotoxicity to neurons should be able to inhibit the growth of glioma cells. Another study indicates that the elevated Ca2+ concentration within glioma cells increases cell activity and thus contributes to the migration of glioma cells. Therefore, Ca2+ channel blocker, such as MK801, can effectively inhibit the proliferation of glioma cells (Ishiuchi et al., 2002). However, MK801 is an antagonist of NMDA receptors, and will result in serious side effects when administered to patients.
Chuanxiong, a Chinese herbal medicine, is generally used in the treatment of brain vascular or cardiovascular embolism. Tetramethylpyrazine (TMP) is one of the major components in chuanxiong. Most of the TMP-related researches focus on the cardiovascular system. Relevant studies indicate that TMP can inhibit the increase of concentration of calcium (Pang et al., 1996). As to the effect of TMP on neurons, we find TMP can reduce the excitotoxicity to neurons induced by kainate through protecting mitochondria and inhibiting free radical generation (Shih et al, 2002). It has also been reported that TMP can cross the blood-brain barrier (Tsai and Liang, 2001).
According to the results of above researches, we suggest that TMP may as a potent medicine in the treatment of brain tumor. In this study, we demonstrate the treatment effects of TMP to C6 glioma and elucidate the underlying mechanisms. In vitro studies, the cultured C6 glioma cells were treated with 50 μM, 100 μM, 200 μM, or 400 μM TMP for 24 hours. Our results shown that 400 μM TMP effectively reduced the ratio of cells in the G2/M phase, implying inhibition of replication of the glioma cells , 200 μM and 400 μM TMP resulted in glioma cell damage, and 50 μM to 400 μM TMP all effectively reduced Ca2+ concentration within glioma cells resulted from the treatment with glutamate. In neuron and glioma cell coculture studies, the ratio of neuronal damage was elevated and the cell number of glioma cells was increased to 140%, moreover, 24 h pretreatment with 12.5 - 100 μM TMP does significantly protect neurons from glioma cells-induced damage. 106 glioma cells were injected into the frontal cortex of the left brain of the rats. The rats of the control group did not receive any treatment following the implantation of glioma cells. The rats of the TMP group received TMP treatment (0.4 mg, subcutaneous injection, and twice a day) from the 8th day following the implantation. The survival days of rats of the two groups were recorded. The results of this experiment showed that TMP could effectively extend the survival time of rats suffering from glioma, and inhibit the size and the activity of glioma in rats.
These findings support a new approach for therapy of brain tumor, based on protecting the neuron from excitotoxicity by glioma cells and inhibiting the activity of glioma cells.
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