白癜风皮损组织中黑素细胞谱系特异性标记基因检测

Detection of Specific Melanocyte Lineage Genes in Vitiligo Lesion Tissues Using RT-PCR Technique

目的针尖皮肤切削术结合逆转录聚合酶链式反应(RT-PCR)技术检测白癜风皮损组织中黑素细胞谱系特异性标记基因的表达,旨在预测治疗后白癜风皮损出现毛囊复色的可能性。方法选取6例就诊于武汉大学人民医院皮肤科静止期白癜风患者和4例健康志愿者,用针尖皮肤切削术对受试者白斑的中心、近边缘和周边"正常皮肤"处活检,抽提组织总RNA,用RT-PCR技术测定标本中多巴色素异构酶(Dct),酪氨酸酶(Tyr)和管家基因β-肌动蛋白(ACTB)mRNA的表达。结果 (1)用针尖皮肤切削术获取不同大小(3 mg和7 mg)的组织标本并与负压吸疱法采集的表皮片进行比较,结果显示针头切削术获取7 mg组织,经研磨异硫氰酸胍-苯酚(Trizol)法抽提总RNA,能在正常皮肤组织中检测到3种基因表达。同时对10例针尖皮肤切削术后的局部伤口愈合进行了追踪随访,1个月后10例均正常愈合,未见瘢痕形成。(2)白癜风皮损黑素细胞谱系特异性标记基因检测:皮损中心区检测出3种模式,即Dct+Tyr-ACTB+,Dct-Tyr-ACTB+和Dct+Tyr+ACTB+。对1例检测结果"Dct+Tyr-ACTB+"的患者施以308 nm准分子光照射2次,皮损即出现毛囊复色。结论针尖皮肤切削术结合RT-PCR技术检测白癜风皮损组织中黑素细胞谱系标记基因表达,是一种有价值的预测白癜风皮损可能出现毛囊复色的微创准确方法。

Objective To detect the expression of specific melanocyte lineage genes in vitiligo lesion tissues using RT-PCR technique combined with a needle biopsy, aming at predicting the occurrence of perifollicular repigmentation in the depigmentary macules. Methods Six stable vitiligo patients and four healthy volunteers were recruited from department of dermatology, Renmin hospital of Wuhan university. Total RNA was extracted from the skin tissues in the near-periphery and center of depigmentary macules as well as the adjacent ＂normal＂ skin that were taken by a needle biopsy. The expression of dopachrome tautomerase （Det）, tyrosinase （Tyr）, and actin-beta （ACTB） gene were detected by a routin RT-PCR technique. Results （1） Three or seven miligram of skin tissue was taken from healthy skin by a needle biopsy and was then used for total RNA extraction by Trizol reagent. The resuhs showed that RNA extracted from seven mg skin tissues was sufficient to amplify three genes to compare with those from three mg tissues and one suction skin sheet. （2） Three gene expression profiles were observed in the center of vitiligous lesions, i.e., Dct＋Tyr-ACTB＋, Dct-Tyr-ACTB＋, and Dct＋Tyr＋ACTB＋. Five-month followup indicated that perifollicular repigmentation was efficaciously induced by 308 nm eximer light radiation in one patient who had a gene expression profile of Dct＋Tyr-ACTB＋. Conclusion Needle biopsy combined with RT-PCR can be used as a minor traumatic and reliable method to detect the expression profiles of specific melanocyte lineage genes, which may has the potential to predict the occurrence of perifollicular repigmentation in vitiligo.