中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2009年
24期
1707-1710
,共4页
万静%刘小明%雷铁池%徐世正
萬靜%劉小明%雷鐵池%徐世正
만정%류소명%뢰철지%서세정
异构酶类%氧化性应激%突变%黑素小体
異構酶類%氧化性應激%突變%黑素小體
이구매류%양화성응격%돌변%흑소소체
Isomerases%Oxidative stress%Mutation%Melanosome
目的 研究多巴色素异构酶(Dct)突变对黑素细胞黑素小体成熟和活性氧基(ROS)清除能力的影响.方法 用透射电子显微镜技术和Fontana-Masson嗜银染色观察小鼠Dct突变slaty黑素细胞和野生型melan-a黑素细胞胞内黑素小体发育及嗜银着色的优黑素颗粒分布;用分光光度计法和蛋白印迹技术测定酪氨酸酶活性和酪氨酸酶,酪氨酸酶相关蛋白1(Tyrp-1)和Dct蛋白表达水平;用二氯荧光素标记法测定两种黑素细胞在3 J/cm2长波紫外线(UVA)照射前后胞内ROS水平的变化.结果 透射电子显微镜观察发现slaty黑素细胞胞内缺乏成熟的Ⅳ期黑素小体,Fontana-Masson嗜银染色也证实slaty黑素细胞胞质几乎完全缺乏嗜银染阳性的优黑素颗粒.与melan-a黑素细胞相比,slaty黑素细胞的离心团块颜色变浅,但slaty细胞的酪氨酸酶活性及蛋白质表达水平与melan-a细胞接近.UVA照射前slaty和melan-a细胞胞内的ROS相对荧光强度分别为8.9±0.7和8.9±2.5,但照射后slaty细胞胞内的ROS相对荧光强度急剧增加,与melan-a黑素细胞相比,差异有统计学意义(18.0±0.3比13.6±3.0,P=0.024).结论 Dct突变导致黑素细胞低色素化表型,影响黑素小体的发育成熟和降低细胞对氧化应激的抵抗能力,尤其是在UVA诱导氧化应激存在时更为明显.
目的 研究多巴色素異構酶(Dct)突變對黑素細胞黑素小體成熟和活性氧基(ROS)清除能力的影響.方法 用透射電子顯微鏡技術和Fontana-Masson嗜銀染色觀察小鼠Dct突變slaty黑素細胞和野生型melan-a黑素細胞胞內黑素小體髮育及嗜銀著色的優黑素顆粒分佈;用分光光度計法和蛋白印跡技術測定酪氨痠酶活性和酪氨痠酶,酪氨痠酶相關蛋白1(Tyrp-1)和Dct蛋白錶達水平;用二氯熒光素標記法測定兩種黑素細胞在3 J/cm2長波紫外線(UVA)照射前後胞內ROS水平的變化.結果 透射電子顯微鏡觀察髮現slaty黑素細胞胞內缺乏成熟的Ⅳ期黑素小體,Fontana-Masson嗜銀染色也證實slaty黑素細胞胞質幾乎完全缺乏嗜銀染暘性的優黑素顆粒.與melan-a黑素細胞相比,slaty黑素細胞的離心糰塊顏色變淺,但slaty細胞的酪氨痠酶活性及蛋白質錶達水平與melan-a細胞接近.UVA照射前slaty和melan-a細胞胞內的ROS相對熒光彊度分彆為8.9±0.7和8.9±2.5,但照射後slaty細胞胞內的ROS相對熒光彊度急劇增加,與melan-a黑素細胞相比,差異有統計學意義(18.0±0.3比13.6±3.0,P=0.024).結論 Dct突變導緻黑素細胞低色素化錶型,影響黑素小體的髮育成熟和降低細胞對氧化應激的牴抗能力,尤其是在UVA誘導氧化應激存在時更為明顯.
목적 연구다파색소이구매(Dct)돌변대흑소세포흑소소체성숙화활성양기(ROS)청제능력적영향.방법 용투사전자현미경기술화Fontana-Masson기은염색관찰소서Dct돌변slaty흑소세포화야생형melan-a흑소세포포내흑소소체발육급기은착색적우흑소과립분포;용분광광도계법화단백인적기술측정락안산매활성화락안산매,락안산매상관단백1(Tyrp-1)화Dct단백표체수평;용이록형광소표기법측정량충흑소세포재3 J/cm2장파자외선(UVA)조사전후포내ROS수평적변화.결과 투사전자현미경관찰발현slaty흑소세포포내결핍성숙적Ⅳ기흑소소체,Fontana-Masson기은염색야증실slaty흑소세포포질궤호완전결핍기은염양성적우흑소과립.여melan-a흑소세포상비,slaty흑소세포적리심단괴안색변천,단slaty세포적락안산매활성급단백질표체수평여melan-a세포접근.UVA조사전slaty화melan-a세포포내적ROS상대형광강도분별위8.9±0.7화8.9±2.5,단조사후slaty세포포내적ROS상대형광강도급극증가,여melan-a흑소세포상비,차이유통계학의의(18.0±0.3비13.6±3.0,P=0.024).결론 Dct돌변도치흑소세포저색소화표형,영향흑소소체적발육성숙화강저세포대양화응격적저항능력,우기시재UVA유도양화응격존재시경위명현.
Objective To investigate whether the mutation in dopachrome tautomerase (Dct) affects melanosome maturation and anti-oxidative potential in cultured melanocytes (MCs). Methods Slaty and melan-a MCs were derived from the skins of neonatal DctSlt and C57 BI/6J mice respectively. Their detailed melanosome structures were examined with a transmission electron microscopy (TEM) and their eu-melanin granules characterized by Fontana-Masson staining. Furthermore, the tyrosinase activity and three melanogenic proteins, I.e. , tyrosinase, tyrosinase-related protein 1 and Dct, were also measured with a spectrophotometery method or Western blot assay. The level of intraceilular reactive oxygen species (ROS) was monitored by 2, 7-dichlorofluorescin diacetate (DCF-DA) labeling. Results Mature stage Ⅳ melanosomes markedly decreased in slaty MCs under TEM. The brownish granules stained with Fontana-Masson silver method were far less in slaty MCs than in melan-a MCs. The cell pellet of slaty MCs was white in color, but the similarities between slaty and melan-a were found in tyrosinase activity and its protein expression. The relative intensity of DCF fluorescence was 8.9±0.7 for slaty melanocytes versus 8.9±2.5 for melan-a melanocytes prior to UVA irradiation, but an abrupt ROS production was merely observed in slaty MCs (18.0±0.3) other than in melan-a MCs (13.6±0.3) after UVA exposure. There was statistical difference between these two cell lines in ROS level upon UVA irradiation (P=0.024). Conclusion The mutation in Det causes hypo-pigmented phenotype in cultured slaty MCs, inhibits melanosome maturation and decreases anti-oxidative capacity especially in the presence of UVA-induced oxidative stress.
