Guan Cui-ping, Lin Fu-quan, Hong Wei-song, Fu Li-fang, Zhou Miao-ni, Xu Wen, Xu Ai-e
Department of Dermatology, Third People's Hospital of Hangzhou, Anhui Medical University, Anhui Medical University, Hangzhou 310009, China.
Zhonghua Yi Xue Za Zhi. 2010 Apr 27;90(16):1126-30.
To investigate the roles of InnVit (FBX011) gene in melanocytes by detecting the expression of InnVit gene in vitiligo and analyzing the impact of InnVit gene on morphology of endoplasmic reticulum (ER) and the tyrosinase export from ER.
The lesion tissues and the donor tissues were collected from 10 vitiligo patients to examine the InnVit gene expression by immunohistochemistry. Synthesized specific siRNA and constructed plasmid P3XF-P120 were separately transfected into cells for the silence and over-expression of InnVit gene with lipofectamine(TM) 2000. The untreated cells were used as control. Morphology of ER of cells from the above three groups was observed under electron microscope. Co-localization of tyrosinase and calreticulin was identified by confocal laser scanning microscopy. InnVit, tyrosinase and calreticulin were examined by Western blot.
In vitiligo patients, the expression of InnVit gene in the lesions was markedly lower than that in the donor tissues. The normal morphology of ER was found in the untreated and the plasmid groups whereas inflated ER was shown in siRNA group. And the relative inflation rate in siRNA group (1.97 +/- 0.48) was higher than that in the untreated group (1.28 +/- 0.09) and plasmid group (1.24 +/- 0.13) (both P = 0.001). In the untreated and the plasmid groups, tyrosinase was expressed beyond the scope marked by ER marker protein calreticulin partly, but co-localized with calreticulin in ER in the siRNA group. Western blot showed that, contrast to the untreated group (0.320 +/- 0.020), a lower expression level of InnVit in the siRNA group (0.030 +/- 0.004, P = 0.001) and a higher expression of InnVit in the plasmid group were shown (0.710 +/- 0.040, P = 0.001). No significant difference about the expression level of calreticulin was observed among the three groups (P > 0.05). As compared with the untreated group (0.350 +/- 0.030), a higher tyrosinase level in the siRNA group (1.040 +/- 0.060, P = 0.001) and in the plasmid group (0.720 +/- 0.030, P = 0.001) was found. And the former was higher than the latter (P = 0.001).
A lower expression of InnVit is observed in the lesion tissues than in the donor tissues from vitiligo patients. The InnVit gene can have an impact on the morphology of ER and tyrosinase export from ER. And it may further affect the function of melanocytes.
通过检测白癜风患者中InnVit（FBX011）基因的表达，分析InnVit基因对内质网（ER）形态及酪氨酸酶从内质网输出的影响，探讨InnVit基因在黑素细胞中的作用。
收集10例白癜风患者的皮损组织和供皮组织，采用免疫组织化学法检测InnVit基因表达。合成特异性小干扰RNA（siRNA）并构建质粒P3XF - P120，分别用脂质体2000转染细胞，使InnVit基因沉默和过表达，未处理细胞作为对照。电镜下观察上述三组细胞内质网形态。通过共聚焦激光扫描显微镜鉴定酪氨酸酶与钙网蛋白的共定位。采用蛋白质免疫印迹法检测InnVit、酪氨酸酶和钙网蛋白。
白癜风患者皮损中InnVit基因表达明显低于供皮组织。未处理组和质粒组内质网形态正常，而siRNA组内质网扩张。siRNA组相对扩张率（1.97±0.48）高于未处理组（1.28±0.09）和质粒组（1.24±0.13）（均P = 0.001）。未处理组和质粒组中，酪氨酸酶部分表达于内质网标记蛋白钙网蛋白标记范围之外，但在siRNA组中与钙网蛋白在内质网中共定位。蛋白质免疫印迹法显示，与未处理组（0.320±0.020）相比，siRNA组中InnVit表达水平较低（0.030±0.004，P = 0.001），质粒组中InnVit表达较高（0.710±0.040，P = 0.001）。三组间钙网蛋白表达水平差异无统计学意义（P＞0.05）。与未处理组（0.350±0.030）相比，siRNA组（1.040±0.060，P = 0.001）和质粒组（0.720±0.030，P = 0.001）酪氨酸酶水平较高，且前者高于后者（P = 0.001）。
白癜风患者皮损组织中InnVit表达低于供皮组织。InnVit基因可影响内质网形态及酪氨酸酶从内质网的输出，进而可能影响黑素细胞功能。
