臺灣博碩士論文加值系統

基質金屬蛋白酶(matrix metalloproteinases，MMPs)是調控神經膠瘤細胞(glioma cells)入侵能力的重要因子之ㄧ。Tanshinone IIA是從一種中藥草-丹蔘(Danshen，Radix Salvia miltiorhiza Bunge)中所純化出來的一種活性成分，對抑制腫瘤的生長和入侵有相當好的效果。本論文利用C6 glioma cells探討tanshinone IIA是否會抑制酯多醣(lipopolysaccharide；LPS)和 PMA (phorbol myristate acetate)誘導MMP-9的表現。實驗結果發現，LPS和PMA處理C6 glioma cells會增加MMP-9的蛋白量和活性，而先以tanshinone IIA處理細胞，則會抑制MMP-9的蛋白量和活性。我們也發現tanshinone IIA會增加細胞中血基質氧化酵素-1(heme oxygenase-1；HO-1)的表現，但是預先以活性氧自由基(reactive oxygen species；ROS)的抑制劑l-NAC (l-N-acetylcysteine) 和PI 3-K抑制劑(LY294002)處理細胞，則皆會抑制tanshinone IIA所誘導的HO-1表現。Tanshinone IIA也會增加Akt/PKB的磷酸化，而且tanshinone IIA所增加的Akt/PKB磷酸化會被l-NAC所減少。接下來以SnPP (tin protoporphyrin)去抑制HO-1表現，發現可以減輕tanshinone IIA抑制MMP-9的蛋白量和活性，顯示HO-1在此抑制效應中扮演關鍵的角色。因為HO-1可催化一氧化碳(CO)的產生，而以CO donor (tricarbonyl dichlororuthenium (Ⅱ) dimer)處理細胞，則可以抑制誘導型一氧化碳合成酶(iNOS)和MMP-9的表現，反之，以CO清除劑血紅素(hemoglobin)處理細胞，則會減輕tanshinone IIA對MMP-9的抑制作用。由於NOS的抑制劑l-NAME會抑制LPS和PMA誘導MMP-9的蛋白量和活性，很可能tanshinone IIA是透過誘導HO-1表現而達到抑制MMP-9的效果，使HO-1催化CO產生，導致iNOS被抑制，進而減少MMP-9的蛋白量和活性。

Matrix metalloproteinases (MMPs) have been implicated as important factors in the control of the invasive capability of glioma cells. Tanshinone IIA, an active ingredient purified from the Chinese herb Danshen (Radix Salvia miltiorhiza Bunge), is known to exhibit potent effects on tumor progression and invasion. In the present study, we investigated whether tanshinone IIA inhibits LPS (lipopolysaccharide) and PMA (phorbol 12-myristate 13-acetate)-induced MMP-9 gene expression in C6 glioma cells. Treatment of C6 glioma cells with LPS and PMA increased the MMP-9 expression and the expression was inhibited by pretreatment of cells with tanshinone IIA at a concentration that is not toxic to C6 glioma cells. Incubation of C6 glioma cells with tanshinone IIA increased heme oxygenase-1 (HO-1) expression, which was inhibited by pretreatment of cells with l-N-acetylcysteine (l-NAC) prior to addition of tanshinone IIA, suggesting reactive oxygen species (ROS) are involved. Inhibition of PI 3-K/Akt pathway by LY294002 or wortmannin reduced the HO-1 protein expression by tanshinone IIA. In agreement, treatment of cells with tanshinone IIA increased phosphorylation of Akt/PKB, which was attenuated by l-Nac suggesting activation of PI3-kinase–Akt is secondary to ROS production. The inhibition of MMP-9 expression by tanshinone IIA was reversed by tin protoporphyrin (SnPP) suggesting tanshinone IIA may exert this inhibitory effect through HO-1. Increase of HO-1 expression catalyzes carbon monoxide (CO) production. Addition of CO donor mimicked the tanshinone IIA effect on suppressing inducible nitrite oxygen synthease (iNOS) and MMP-9 expression. Scavenge of CO by hemoglobin (Hb) reversed the inhibition due to tanshinone IIA. Inhibition of nitric oxide (NO) production by l-NAME inhibited MMP-9 expression due to tanshinone IIA. Taken together, these results suggest that tanshinone IIA exerts its inhibitory effect through induction of HO-1 expression. HO-1 catalyzes the formation of CO, which in turn inhibit iNOS induction. Inhibition of iNOS expression subsequently reduces the MMP-9 protein level and activity.

目錄

中文摘要 I
Abstract III
目錄 V
圖目錄 VII
縮寫表 X

壹、 緒論 1
一、 腦瘤(Brain Tumor) 2
二、 丹蔘酮(Tanshinones) 3
三、 血紅素氧化酵素(Heme Oxygenases；HOs) 5
四、 活性氧自由基(Reactive Oxygen Species；ROS) 6
五、 Phosphatidylinositol 3-kinase (PI 3-K) pathway 7
六、 Nuclear factor-erythroid 2 (NF-E2) 8
七、 一氧化氮合成酶(Nitric Oxide Synthase；NOS) 9
八、 基質金屬蛋白酶(Matrix Metalloproteinases；MMPs) 10

貳、 實驗材料與方法 13
一、 實驗材料 13
1、 藥品試劑 13
2、 常用儀器 15
3、 常用溶液 17
4、 電泳酵素分析法(gelatine zymography) buffer 18
5、 C6神經膠瘤細胞株(C6 glioma cell line) 19
6、 中草藥tanshinone IIA(化學結構式如下) 19

二、 實驗方法 20
1、 C6神經膠瘤細胞株(C6 glioma cell line)繼代培養 20
2、 細胞存活率試驗(cell viability) 20
a. 細胞計數法： 20
b. PI染色法： 21
3、 細胞蛋白質的測定 22
4、 流式細胞儀分析 23
a. 細胞處理 23
b. 活性氧自由族(ROS)的測量 23
5、 細胞RNA萃取(preparation of cell RNA) 24
6、 聚合酵素鏈鎖反應(reverse transcriptase-polymerase chain reaction；RT-PCR) 25
7、 電泳酵素分析法 27
8、 統計分析 27

參、 實驗結果 28
一、 Tanshinone IIA影響LPS和PMA誘導C6 glioma cells MMP-9的量和活性 28
二、 Tanshinone IIA誘導C6 glioma cells HO-1的基因表現 28
三、 ROS在tanshinone IIA誘導HO-1表現中扮演的角色 29
四、 Tanshinone IIA誘導HO-1表現的訊息傳遞路徑 30
五、 HO-1在tanshinone IIA抑制LPS和PMA所誘導的MMP-9中所扮演的角色 32
六、 iNOS在tanshinone IIA抑制MMP-9所扮演的角色 33

肆、 討論 34

伍、 參考文獻 38
圖目錄

Fig. 1. Effects of tanshinone IIA on cell viability in C6 glioma cells. 46

Fig. 2. Effects of tanshinone IIA on cell cycle progression in C6 glioma cells. 47

Fig. 3. Tanshinone IIA inhibited LPS and PMA-induced iNOS and MMP-9 expression. 48

Fig. 4. Tanshinone IIA induced a dose dependent increase in HO-1 protein expression in C6 glioma cells. 49

Fig. 5. Tanshinone IIA induced a time dependent increase in HO-1 protein expression in C6 glioma cells. 50

Fig. 6. Effects of actinomycin D (Act D) and cycloheximide (CHX) on tanshinone IIA-induced HO-1 expression in C6 glioma cells. 51

Fig. 7. Effects of actinomycin D (Act D) and cycloheximide (CHX) on tanshinone IIA-induced HO-1 mRNA expression in C6 glioma cells. 52

Fig. 8. Tanshinone IIA induced a dose dependent increase in HO-1 mRNA expression in C6 glioma cells. 53

Fig. 9. Tanshinone IIA induced a time dependent increase in HO-1 mRNA expression in C6 glioma cells. 54

Fig. 10. Effects of l-NAC and BSO on tanshinone IIA-induced HO-1 expression in C6 glioma cells. 55

Fig. 11. Tanshinone IIA induced ROS generation in C6 glioma cells. 56


Fig. 12. Effects of mitochondrial complex I inhibitor on tanshinone IIA-induced HO-1 expression in C6 glioma cells. 57

Fig. 13. Effects of mitochondrial complex III inhibitor on tanshinone IIA-induced HO-1 expression in C6 glioma cells. 58

Fig. 14. Effects of mitochondrial complex IV inhibitor on tanshinone IIA-induced HO-1 expression in C6 glioma cells. 59

Fig. 15. Effects of PI 3-K inhibitor on tanshinone IIA-induced HO-1 expression in C6 glioma cells. 60

Fig. 16. Effects of PI 3-K inhibitor on tanshinone IIA-induced HO-1 expression in C6 glioma cells. 61

Fig. 17. Tanshinone IIA induced PDK-1 phosphorylation in C6 glioma cells. 62

Fig. 18. Tanshinone IIA induced Akt phosphorylation in C6 glioma cells. 63

Fig. 19. Effects of l-NAC on tanshinone IIA-induced Akt phosphorylation in C6 glioma cells. 64

Fig. 20. Effects of p38 MAPK inhibitor on tanshinone IIA-induced HO-1 expression in C6 glioma cells. 65

Fig. 21. Effects of MAP kinase and NF-B inhibitors on tanshinone IIA-induced HO-1 expression in C6 glioma cells. 66

Fig. 22. HO-1 mediates the inhibition of LPS and PMA-induced MMP-9 protein and activity in C6 glioma cells following treatment with tanshinone IIA. 67

Fig. 23. CO donor inhibited LPS and PMA-induced MMP-9 protein and activity. 68


Fig. 24. CO mediates the inhibition of LPS and PMA-induced MMP-9 protein and activity in C6 glioma cells following treatment with tanshinone IIA. 69

Fig. 25. HO-1 mediates the inhibition of LPS and PMA-induced iNOS expression in C6 glioma cells following treatment with tanshinone IIA. 70

Fig. 26. NO mediates the inhibition of LPS and PMA-induced MMP-9 protein and activity in C6 glioma cells. 71

Fig. 27. Schematic illustration of signal transduction mechanism of tanshinone IIA-inhibited MMP-9 expression in C6 glioma cells. 72

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