臺灣博碩士論文加值系統

癌細胞的轉移擴散是癌症病人致死的主要原因之一，其中血管新生 (angiogenesis)為腫瘤生長及癌細胞轉移中的重要步驟，受到介白素 (Interleukin, IL)-12、IL-8和血管內皮生長因子 (Vascular endothelial growth factor, VEGF)等因子的調控。 樟芝 (Antrodia cinnamomea)為台灣特有的蕈類，研究指出樟芝多醣體 (polysaccharides isolated from Antrodia cinnamomea, AC-PS)在動物及細胞試驗中可透過調節免疫系統而抑制癌細胞生長，但是否能抑制癌細胞轉移及其作用機制目前並不清楚。 因此，本研究探討樟芝菌絲體萃取之多醣體可否經由免疫調節抑制癌細胞轉移中的血管新生作用，並進一步釐清其作用的分子量範圍與機制。 實驗方法：利用膜過濾將熱水萃取之AC-PS區分成四個分子量範圍 (＜5 kD、5~30 kD、30~100 kD、＞100 kD)，並與人類周邊血液分離出的單核細胞 (Mononuclear cells, MNC)培養三天後，分析條件培養液 (conditioned medium)中IL-12、IL-8、γ型干擾素 (Interferon-γ, INF-γ)及前列腺素E2 (Prostaglandin E2, PGE2)的分泌量。 結果顯示：分子量＞100 kD的AC-PS可顯著促進MNC分泌IL-12 (p＜0.00001)及IFN-γ (p＜0.0001)的分泌。 當濃度為100 μg/ml時，分子量＞100 kD的AC-PS可使IL-12與IFN-γ之分泌量上升約27.7及3.3倍，而與正控制組PHA促進的效果相當，且呈現劑量關係。 接著將培養MNC後的條件培養液 (AC-PS-MNC-CM)與人類血癌細胞 (human leukemia cells, HL-60)培養三天，發現在分子量＞100 kD的組別，在劑量為100 μg/ml時，與控制組相比可顯著抑制38.9 %的VEGF分泌 (p＜0.05)，且其效果較PHA組顯著。 最後以AC-PS-MNC-CM與人類臍靜脈內皮細胞 (human umbilical vein endothelial cells, HUVECs)培養後，同樣發現在分子量＞100 kD的組別可抑制其脈絡生成 (tube formation)，且其抗血管新生的效果也隨劑量上升而增強；但其他分子量範圍的組別中則無明顯的作用。 本研究證明，分子量＞100 kD之AC-PS可藉由調節細胞激素分泌來抑制HUVECs血管新生作用，因而可能具有抑制癌細胞轉移的能力，且其抗血管新生機制可能是(1)透過刺激MNC分泌IL-12和Th1型細胞激素IFN-γ的分泌；(2)透過活化的IL-12直接產生作用；和(3)透過活化的IL-12抑制癌細胞VEGF的分泌。

Metastasis is one of the major causes of cancer-associated mortality, and angiogenesis is a key step in the growth and metastasis of tumor cells. Angiogenesis is regulated by several angiogenic or angiostatic factors including vascular endotheilial growth factor (VEGF), Interleukin(IL)-8, IL-12, and others. Antrodia (A.) cinnamomea is a species known to be available only in Taiwan. Polysaccharides isolated from A. cinnamomea mycelia (AC-PS) elicit its anti-tumor effect by activating host immune response in both in vitro and in vivo models. However, there are few reports on the anti-angiogenic activity of AC-PS, and little is known about its mechanisms of action. In this study, we determined the anti-angiogenic activity of AC-PS in vitro. Furthermore, we investigated the anti-angiogenic mechanism of AC-PS in different molecular-weight (MW) fractions of AC-PS. The polysaccharides were isolated from the mycelia of A. cinnamomea using ethanol precipitation methods and fractionated into four MW fractions (＜5 kD, 5~30 kD, 30~100 kD, and ＞100 kD). We first pre-incubated human mononuclear cells (MNC) with different MW fractions of AC-PS to investigate these immunomodulatory effects. We found that Interleukin-12 (IL-12) and Interferon-γ (IFN-γ) levels in AC-PS-MNC-CM were significantly increased in a dose-dependent manner (p＜0.0001 and p＜0.0001, respectively) in the fraction with MW＞100 kD; at 100 μg/ml, IL-12 and IFN-γ increased 27.7 and 3.3 folds, respectively, as compared to the control.
Then, we pre-incubated human leukemia cells (HL-60) and human umbilical vein endothelial cells (HUVECs) with AC-PS-stimulated-MNC-conditioned medium (AC-PS-MNC-CM) to investigate the anti-angiogenic effect and mechanisms underlying the action of AC-PS. In the fraction with MW＞100 kD, VEGF secretion in HL-60 was significantly decreased in a dose-dependent manner (p＜0.05). Similar phenomenon was observed on Matrigel tube formation of HUVECs (pretreated with AC-PS-MNC-CM) in the fraction with MW＞100 kD but not in other MW fractions.
These results suggeste that AC-PS with MW＞100 kD elicit its anti-angiogenic activities through immunomodulation, and that these effects are attributed to the regulation of release of IL-12 and IFN-γ, the activation by IL-12 directly, and the inhibition of VEGF secretion in tumors mediated by IL-12 from activated mononuclear cells.

目 錄

縮寫表……………………………………………………………………………... VII

中文摘要…………………………………………………………………………. IX

英文摘要…………………………………………………………………………. XI

文獻整理…………………………………………………………………………. 1
一、 癌症與轉移………………………………………………………………... 1
二、 癌症的轉移與血管新生…………………………………………………... 2
三、 血管新生與血管內皮生長因子…………………………………………... 3
四、 癌細胞轉移與基質金屬蛋白水解酵素…………………………………... 5
五、 血液惡性腫瘤簡介………………………………………………………... 6
六、 免疫與癌症………………………………………………………………... 7
七、 細胞激素與血管新生……………………………………………………... 9
八、 細胞激素與VEGF……………………………………………………….... 12
九、 食藥用菇…………………………………………………………………... 13
十、 樟芝………………………………………………………………………... 13
十一、 多醣體介紹…………………………………………………………….. 17
十二、 多醣體的吸收與作用途徑…………………………………………….. 19

研究動機與目的…………………………………………………………………. 22

實驗架構…………………………………………………………………………. 24

材料與方法………………………………………………………………………. 25
一、 樟芝菌絲體………………………………………………………………... 25
二、 實驗試劑…………………………………………………………………... 25
三、 樟芝多醣體萃取與分子量劃分………..…………………………………. 26
四、 多醣體含量分析………………….……………………………………….. 26
五、 中性單醣組成分析………………..………………………………………. 26
六、 活性多醣分析……………………………………………………………... 27
七、 細胞解凍…………………………………………………………………... 28
八、 細胞保存…………………………………………………………………... 28
九、 人類血癌細胞培養………………………………………………………... 29
十、 人類臍靜脈內皮細胞培養…………..……………………………………. 29
十一、 人類單核球細胞的分離及條件培養液的製備………………………. 29
十二、 條件培養液之抗轉移試驗………………………………………..…... 30
十三、 細胞毒性分析…………………………………………………………. 31
十四、 蛋白質定量………………………………………………………...….. 31
十五、 細胞激素之測定………………………………………………...…….. 31
十六、 培養液中VEGF之測定……………………………………...……….. 32
十七、 培養基中MMP-2, -9之測定……………………………...………….. 32
十八、 西方點墨法……………………………………………...…………….. 33
十九、 體外血管新生分析……………………………………...…………….. 35
二十、 統計分析……………………………………………...……………….. 35

結果………………………………………………………………………………. 36
一、 樟芝多醣體成份分析……………….……………………………………. 36
二、 不同分子量的樟芝多醣體對單核球細胞分泌細胞激素之影響……….. 36
三、 樟芝多醣體對MNC細胞內IL-12蛋白表現之影響……………….…... 37
四、 不同分子量的樟芝多醣體對發炎反應之影響……………………….…. 37
五、 樟芝多醣體 (MW＞100 KD)對MNC的細胞毒性…………………….. 38
六、 樟芝多醣條件培養液對HUVECs細胞脈管形成之影響……….……… 38
七、 IL-12對HUVECs細胞脈管形成之影響……………………….……...... 38
八、 樟芝多醣條件培養液對HL-60細胞MMP-2, -9分泌之影響.………… 39
九、 樟芝多醣條件培養液對HL-60細胞分泌VEGF之影響……….…….... 39
十、 直接加入樟芝多醣培養對HL-60細胞分泌VEGF之影響….………… 40
十一、 IL-12對HL-60細胞分泌VEGF之影響…………………….………. 40

討論………………………………………………………………………………. 41
一、 樟芝多醣調節免疫能力之探討…………………………………………. 41
二、 樟芝多醣經由免疫調節而抑制血管新生之機制探討…………………. 43
三、 樟芝多醣免疫調節能力與其成分組成之相關性………………………. 44

結論………………………………………………………………………………. 45

圖表………………………………………………………………………………. 47

文獻整理………………………………………………………………………..... 67




圖 表 目 次

表目次
Table 1. The content of polysaccharides in water extract of mycelia from Antrodia
cinnamomea………………………………………………………………...47

Table 2. The content of different molecular weight in AC-PS………………….........48

Table 3. The content of β-1, 3-glucan in different MW fractions of AC-PS………....49

Table 4. Characteristics of AC-PS in different MW fractions…………………..........50

圖目次
Fig. 1. IL-12 levels in MNC-CM prepared with different MW fractions of AC-PS....51

Fig. 2. IL-8 levels in MNC-CM prepared with different MW fractions of AC-PS…..52

Fig. 3. IFN-γ levels in MNC-CM prepared with different MW fractions of AC-PS…53

Fig. 4. Effects of AC-PS (MW＞100 kD) on intracellular IL-12 expression in MNC.…………………………………………………………………………..54

Fig. 5. PGE2 levels in MNC-CM prepared with different MW fractions of AC-PS....55

Fig. 6. Effect of AC-PS (MW> 100 kD) on the release of LDH……………………..56

Fig. 7. Effects of AC-PS (MW ＜5 kD) on HUVEC capillary tube formation on Matrigel………………………………………………………………………..57

Fig. 8. Effects of AC-PS (MW 5~30 kD) on HUVEC capillary tube formation on Matrigel………………………………………………………………………..58

Fig. 9. Effects of AC-PS (MW 30~100 kD) on HUVEC capillary tube formation on Matrigel………………………………………………………………………..59

Fig. 10. Effects of AC-PS (MW ＞100 kD) on HUVEC capillary tube formation on Matrigel………………………………………………………………………..60

Fig. 11. Effects of IL-12 on HUVEC capillary tube formation on Matrigel…………61

Fig. 12. Effect of AC-PS-MNC-CM on MMP-2 and MMP-9 activities in HL-60 cells…………………………………………………………….……………..62

Fig. 13. Effects of AC-PS-MNC-CM on secreted VEGF in HL-60 cells…………….63

Fig. 14. Effects of AC-PS on secreted VEGF in HL-60……………………………...64

Fig. 15. Effects of IL-12 on secreded VEGF in HL-60 cells………………………...65

Fig.16. Possible mechanisms of AC-PS on inhibited angiogenesis through
immunomodulation…………………………………………………………..66

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