臺灣博碩士論文加值系統

Ｍoscatilin是從蘭科植物石斛中提取出的化合物，許多的研究指出此化合物具有抗腫瘤的活性。Daphnoretin則是Wikstroemiaindica C.A. Meys的一個活性成分，也被證實具有抗癌作用。因此，在我們實驗中，以此兩種天然的抗癌藥物，進行研究。一來探討驗證moscatilin對於人類食道癌的抑制作用以及開發可能的輻射增敏效用。二來開發daphanoretin的免疫調控作用，以daphnoretin處理單核球後，觀察其對樹突細胞的分化與成熟的影響。結果發現，在細胞實驗中，moscatilin對於兩種不同的食道癌細胞都有抑制生長的作用。而且在moscatilin作用後，可見到細胞進行凋亡與分裂風暴。藥物處理後，細胞分布在Sub-G1期和多倍體期的族群也明顯增加。進一步在免疫螢光染色的實驗中觀察到，moscatilin處理後細胞出現多極的細胞分裂和多核的現象，隨後才形成分裂風暴。西方墨點試驗顯示，經moscatilin處理後，癌細胞內的polo-like kinase 1（Plk1）和cyclin B1的表現量增加。另外，我們進一步探討，moscatilin在動物實驗上的作用。Moscatilin確實能抑制移植上小鼠的腫瘤的生長，而且並未導致嚴重的毒性反應。此外，以moscatilin前處理食道癌細胞後，會增加癌細胞對放射治療的敏感度。
而在daphnoretin實驗中，藥物處理單核球後，樹突細胞的回復率下降。成熟的樹突細胞具有較短與較少的樹突。Daphnoretin降低CD1a, CD40, CD83, DC-SIGN, HLA-DR的表現量進而調控樹突細胞的分化與成熟。Daphnoretin 抑制了樹突細胞對於naive CD4+CD45+RA+ T cell的異體刺激活性。機轉上的結果顯示，Daphnoretin可能藉由下調磷酸化c-Jun N-terminal kinase (pJNK)的表現達到此結果。在動物皮膚移植與樹突細胞實驗中，Daphnoretin抑制了急性排斥作用，延長了移植皮膚的存活。
綜合來看，我們的實驗顯示，moscatilin除了具有抑制食道癌生長的效果外，也有輻射增敏的作用。而daphnoretin能調控樹突細胞的分化與成熟來達到免疫調控的目的.因此，我們經由藥物的不同機轉，來研究開發出新的作用，可應用在更多的適應症。

Moscatilin, a bibenzyl derivative from the orchid Dendrobium loddigesii, has been shown to possess anticancer activity. Daphnoretin, an active constituent of Wikstroemiaindica C.A. Meys, has also been shown possessing anti-cancer activity. We examined the effect of moscatilin on human esophageal cancer cells, including squamous cell carcinoma (SCC) and adenocarcinoma (ADC) cells and its possible mechanisms. We examined the effect of daphnoretin on differentiation and maturation of human myeloid dendritic cells (DCs), too. The results showed moscatilin suppressed the growth of both the histological cell lines in a dose- and time-dependent manner. Morphological changes indicative of apoptosis and mitotic catastrophe were observed following moscatilin treatment. The population of cells in the sub-G1 phase and polyploidy phase significantly increased after treatment. Immunofluorescence revealed multipolar mitosis and subsequent multinucleation in moscatilin-treated cells, indicating the development of mitotic catastrophe. Western blot showed a marked increase inexpressions of polo-like kinase 1 and cyclin B1 after exposure to moscatilin. In conclusion, moscatilin inhibits growth and induces apoptosis and mitotic catastrophe in human esophageal SCC- and ADC-derived cell lines, indicating that moscatilin has broad potential against esophageal cancer. We further aimed to validate the in vitro activity and Plk1 expression in vivo following moscatilin treatment and to examine the treatment’s radiosensitizing effect. Moscatilin significantly suppressed tumor growth in mice bearing human esophageal xenografts without affecting body weight, white blood cell counts, or liver and renal function. Moreover, moscatilin pretreatment enhanced CE81T/VGH and BE-3 cell radioresponse in vitro.
In addition, after treatment with daphnoretin (0, 1.1, 3.3, 10 and 30 M) to initiate monocytes, the recovery rate of DCs was reduced in a dose-dependent manner. The mature DCs differentiated in the presence of daphnoretin had fewer and shorter dendrites. Daphnoretin modulated DCs differentiation and maturation in terms of lower expression of CD1a, CD40, CD83, DC-SIGN, HLA-DR. Daphnoretin inhibited the allostimulatory activity of DCs on proliferation of naive CD4+CD45+RA+T cell. On the mitogen-activated protein kinase, daphnoretin down-regulated the lipopolysaccharide-augmented expression of phosphorylated c-Jun N-terminal kinase (pJNK), but not p38 and extracellular signal-regulated kinase 1/2 (ERK1/2). Activation of JNK by anisomycin reversed the effect of daphnoretin on daphnoretin-inhibited pJNK expression and dendrite formation of DCs. In disease model related to maturation of DCs, daphnoretin suppressed the acute rejection of skin allografts in mice.
In conclusion, moscatilin inhibits growth and induces apoptosis and mitotic catastrophe in human esophageal SCC- and ADC-derived cell lines. Moscatilin may also inhibit growth of human esophageal tumors and sensitize esophageal cancer cells to radiation therapy. Daphnoretin modulated differentiation and maturation of DCs toward a state of atypical maturation with impaired allostimulatory function and this effect may go through down-regulation of phosphorylated JNK. Therefore, we perhaps can develop novel indication from natural drugs through different mechanisms.

指導教授推薦書
口試委員會審定書
致謝 iii
中文摘要 iv
英文摘要 vi
目錄 ix
圖目錄 xi
表目錄 xii
CHAPTER 1 INTRODUCTION ..................................................... - 2 - SECTION 1 MOSCATILIN ...............................................................................................................................................- 2 - SECTION 2 DAPHNORETIN ...........................................................................................................................................- 5 -
CHAPTER 2 MATERIALS AND METHODS ............................................ - 8 - SECTION 1 MOSCATILIN ..............................................................................................................................................- 8 - SECTION 2 DAPHNORETIN ........................................................................................................................................ - 16 -
CHAPTER 3 RESULTS......................................................... - 21 - SECTION 1 THE INHIBITION AND RADIOSENSITIZATION OF MOSCATILIN IN ESOPHAGEAL CANCER... - 21 - SECTION 2 THE IMMUNITY MODULATION OF DAPHNORETIN IN DC............................................................ - 40 -
CHAPTER 4 DISCUSSION ...................................................... - 50 - SECTION1 THE INHIBITION AND RADIOSENSITIZATION OF MOSCATILIN .................................................... - 50 -
SECTION 2 THE IMMUNITY MODULATION OF DAPHNORETIN......................................................................... - 54 -
CHAPTER 5 CONCLUSIONS ..................................................... - 57 -
REFERENCES ............................................................... - 58 -

圖目錄
Figure 1 ............................................................................................................... - 22 -
Figure 2 ............................................................................................................... - 24 -
Figure 3 ............................................................................................................... - 25 -
Figure 4 ............................................................................................................... - 29 -
Figure 5 ............................................................................................................... - 32 -
Figure 6 ............................................................................................................... - 33 -
Figure 7 ............................................................................................................... - 34 -
Figure 8 ............................................................................................................... - 36 -
Figure 9 ............................................................................................................... - 37 -
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Figure 11 ............................................................................................................. - 39 -
Figure 12 ............................................................................................................. - 41 -
Figure 13 ............................................................................................................. - 42 -
Figure 14 ............................................................................................................. - 43 -
Figure 15 ............................................................................................................. - 44 -
Figure 16 ............................................................................................................. - 47 -
Figure 17 ............................................................................................................. - 49 -

表目錄
Table 1 - 26 -
Table 2 - 32 -
Table 3 - 36 -

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