PUVA及窄波長紫外線B對黑色素細胞增生及移動之影響

Effects of PUVA and narrow-band UVB on proliferation and migration in melanocytes

吳慶軒(Ching-Shuang Wu)

指導教授 ： 余幸司 ； 陳國熏

高雄醫學大學/醫學院/醫學研究所/博士(2004年)

摘要

尋常性白斑為一種皮膚脫色素性疾病，其罹患率為1-4%。病灶處之功能性黑色素細胞因不明之致病機轉而消失。PUVA光化學療法為臨床上治療尋常性白斑之有效方法，然而詳細的治療機轉仍不甚清楚。因此本章之研究目的為探討PUVA對體外培養之黑色素細胞增生及移動之影響。研究結果顯示，PUVA 對於黑色素細胞在不同細胞外細胞基質蛋白之附著、增生及移動均無明顯的影響，此外PUVA 照射處理黑色素細胞後也無法促進growth factors 之分泌。PUVA 照射處理後之角質細胞上清液並無法對於黑色素細胞之增生與移動產生明顯之影響，而角質細胞經過PUVA照射處理後在黑色素細胞growth factors之分泌也與未照射之對照組無明顯差異。PUVA 照射處理黑色素細胞後其上清液中MMP-2 之表現有明顯增加的趨勢，因此推測臨床上PUVA對白斑病人治療後會刺激黑色素細胞MMP-2 之表現而促進黑色素細胞之移動。由過去的研究得知PUVA可減少黑色素細胞上vitiligo-associated melanocyte antigen (VAMA) 之表現，減少Langerhans 細胞的數目並且增加tyrosinase 之活性，因此綜合我們的研究結果與過去文獻報告，推測PUVA在臨床上之療效應該是製造一個利於黑色素細胞生長的環境而使得黑色素細胞恢復再生。窄波長紫外線B (narrow-band UVB) 是臨床上治療白斑有效的方法，然而這種療法在白斑病灶處造成色素恢復之機轉並未被完全解明。本研究的目的是要探討narrow-band UVB對體外培養之黑色素細胞增生與移動之影響。結果顯示narrow-band UVB照射角質細胞後之上清液能夠增加黑色素細胞數目並且促進細胞 [3H]thymidine uptake。而這些上清液中bFGF與ET-1 之含量明顯增加。bFGF被認為是黑色素細胞之生長刺激素，而ET-1 則能夠刺激黑色素細胞合成DNA，而這種刺激黑色素細胞增生之現象可以被selective endothelin B receptor antagonist (BQ788) 所抑制，顯示ET-1在促進黑色素細胞增生是不可或缺的。此外narrow-band UVB照射能夠促進黑色素細胞之移動，FAK (focal adhesion kinase) 在細胞移動上扮演關鍵角色，而我們的研究結果顯示黑色素細胞表面磷酸化FAK (p125FAK) 之表現會因為narrow-band UVB之照射而增加。我們的研究結果顯示narrow-band UVB照射後黑色素細胞上清液中MMP-2之表現增加，而narrow-band UVB照射黑色素細胞所促進之移動增加可以因為加入p125FAK inhibitor (herbimycin A) 或 MMP-2 inhibitor (GM6001) 而明顯被抑制，顯示p125FAK 與MMP-2 在narrow-band UVB照射黑色素細胞所促進之移動增加上扮演重要的角色。我們的研究結果提供了臨床上白斑病人以narrow-band UVB治療成效之理論基礎。

關鍵字

窄波長紫外線B ； 黑色素細胞 ； 增生 ； 移動

並列摘要

Functional melanocytes in patients with vitiligo vulgaris disappear from involved skin by a mechanism(s) that has not yet been identified. PUVA is an effective treatment for vitiligo. However, the mechanisms of PUVA in repigmentation are not thoroughly clarified. The purpose of our study was to investigate the effects of PUVA treatment on proliferation and migration of melanocytes in vitro. The results revealed that PUVA treatment demonstrated no significant effects on the attachment of ECMs、proliferation and migration in melanocytes. No growth factors were detected in supernatants derived from PUVA treated melanocytes. The release of melanocyte growth factors in supernatants derived from PUVA treated keratinocytes were not different from control groups. The supernatants derived from PUVA treated keratinocytes demonstrated no significant effects on melanocyte proliferation and migration. However, a significant increase in expression of MMP-2 in supernatants derived from PUVA treated melanocytes was noticed. It is reasonable to proposed that the increased expression of MMP-2 in PUVA treated melanocytes supernatants could directly facilitate the melanocyte migration during the process of repigmentation in vivo. Previous results showed that PUVA treatment would suppress the expression of vitiligo-associated melanocyte antigen (VAMA) on melanocytes，deplete epidermal Langerhans cells in vivo and stimulate tyrosinase activity. We proposed that PUVA treatment may create a favorable milieu for the sustaining of melanocytes instead of stimulating the regrowth of melanocytes. Narrow-band UVB radiation is an effective treatment for vitiligo vulgaris. However, the mechanisms of narrow-band UVB in inducing repigmentation of vitiligo lesions are not thoroughly clarified. The purpose of our study was to investigate the effects of narrow-band UVB irradiation on melanocyte proliferation and migration in vitro. Our results showed that the cell counts as well as [3H]thymidine uptake of melanocytes were significantly enhanced by narrow-band UVB irradiated keratinocyte supernatants. In these supernatants, a significant increase in basic fibroblast growth factor (bFGF) and endothelin-1 (ET-1) release was noticed. Basic fibroblast growth factor is a natural mitogen for melanocytes, whereas ET-1 can stimulate DNA synthesis in melanocytes. This stimulatory effect of melanocyte proliferation by supernatants derived from narrow-band UVB irradiated keratinocytes was significantly reduced by a selective endothelin B receptor antagonist (BQ788), suggesting an essential role of ET-1 on melanocyte proliferation. Our results of time-lapse microphotography revealed a stimulatory effect of narrow-band UVB irradiation on melanocyte migration. Focal adhesion kinase (FAK) plays a pivotal role in cell migration. Phosphorylated focal adhesion kinase (p125FAK) expression on melanocyte was enhanced by narrow-band UVB irradiation. In this study, narrow-band UVB irradiation stimulated a significant increase in MMP-2 activity in melanocyte supernatant. Narrow-band UVB irradiation induced-migration of melanocytes was significantly annihilated by addition of p125FAK inhibitor (herbimycin A) or MMP-2 inhibitor (GM6001). These results suggest that p125FAK and MMP-2 activity play important roles in narrow-band UVB induced-migration of melanocytes. Our results provide a theoretical basis for the effectiveness of narrow-band UVB irradiation in treating vitiligo.

並列關鍵字

PUVA ； narrow-band UVB ； melanocyte ； proliferation ； migration

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