【作者】 林福全；

【导师】 胡秀芳；

【作者基本信息】 浙江理工大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 白癜风是一种色素脱失性皮肤病,以表皮、粘膜和毛发的黑素细胞脱失形成的白斑、灰白发为主要症状,其发病机制存在自身免疫学、氧化应激学、神经学以及遗传学等多种假说。近年来,国内外大量研究表明白癜风的发病与遗传因素密切相关,可能是一种多基因遗传病。据报道InnVit基因是与白癜风患者的黑素细胞表达下调相关的新基因。通过序列比对发现InnVit基因为缺少81bp内含子的FBXO11基因。为了探讨InnVit基因在黑素细胞中的生物学功能,明确InnVit基因在白癜风中的作用,本论文构建了沉默InnVit基因的siRNA及InnVit基因过表达载体p3XF-P120,研究InnVit基因的抑制和过表达对黑素细胞生物学活性及酪氨酸酶输出的影响。论文通过设计构建了人工合成的特异性siRNA,同时构建了该基因的过表达质粒,同时分别转染黑素细胞,从而研究InnVit基因抑制和过表达对黑素细胞生物学功能及黑素细胞酪氨酸酶表达及内质网输出的的影响。首先,检测了InnVit基因mRNA和蛋白的表达,siRNA的抑制作用显著降低了InnVit基因mRNA和蛋白的表达量,p3XF-P120质粒的过表达显著增加了InnVit基因mRNA和蛋白的表达量。其次,InnVit基因抑制和过表达对黑素细胞增殖、凋亡及迁移相关生物学功能的影响。采用siRNA抑制和质粒载体过表达后,InnVit基因的过表达促进了细胞增殖,抑制细胞凋亡;InnVit基因的抑制使黑素细胞会出现从G0/G1期到S期的细胞周期停滞现象,极大地降低了细胞增殖和存活的能力,同时显著增加黑素细胞的早期凋亡水平。细胞迁移能力及酶活性检测表明,siRNA抑制和过表达InnVit基因对细胞迁移力及2金属蛋白酶MMP9和MMP的表达没有影响。推测,InnVit基因的抑制主要通过促进细胞凋亡和细胞周期停滞而降低黑素细胞的存活和增殖能力。最后,研究了InnVit基因对黑素细胞内质网膨胀与酪氨酸酶的相关性。扫描电镜观察表明,siRNA抑制的黑素细胞内质网有明显的膨胀扩大现象,其酪氨酸酶蛋白表达存在极显著的增加。细胞免疫荧光共定位了酪氨酸酶和内质网标记钙网蛋白,发现抑制组细胞的酪氨酸酶大部分停留在内质网中,表明其酪氨酸酶的内质网输出发生阻滞,可见抑制InnVit基因表达影响了黑素细胞的酪氨酸酶从内质网输出,导致了细胞内质网膨胀,从而导致细胞生物学活性异常。以上研究表明,本研究为InnVit基因影响黑素细胞的增殖和凋亡能力提供了有力的证据,并揭示了白癜风黑素细胞酪氨酸酶的内质网功能性输出障碍的可能机制。更多还原

【Abstract】 Vitiligo is a common autoimmune, frequent family clustering depigmentary disorder of skin that results from destruction melanocytes. To date, the pathogenesis of vitiligo is unknown although there are many theories about its etiology, including autoimmune, neural and genetic hypotheses. In recent years, genetic contributions were revealed as the most important causative factor of vitiligo. Moreover, scientists holding the other hypotheses of vitoligo began to seek supporting evidence from genetics.InnVit, a novel gene, was reportedly downregulated in vitiligo melanocytes. Sequence analysis revealed that the InnVit gene lacking its intron is the FBXO11 gene. To analyze the effect of InnVit gene on MC biological behavior further investigates molecular mechanisms of InnVit gene on Tyrosinase. We designed and synthesized siRNA and expression vectors for the InnVit gene, Here, we further investigate the effects of inhibition and over-expression of InnVit gene on the biological behavior of melanocytes, and on the export of Tyrosinase from ER.First, transfection efficiency was estimated by RT-PCR and western blot analysis at post-transfection, Silencing of InnVit gene remarkly decreased the expression of mRNA and protein of InnVit,while over-expression of InnVit gene increased the expression of mRNA and protein of InnVit .Second,We detected biological behavior of melanocytes on cell proliferation, apoptosis, transwell and melanin synthesis. The results demonstrated that the silencing of InnVit gene inhibited cell proliferation and induced cell apoptosis efficiently, while the over-expression of InnVit gene could promote cell proliferation and suppress cell apoptosis. Silencing of InnVit gene resulted in a decreased ability to progress from G0/G1 phase to S phase, eventually inhibiting cell proliferation, which was confirmed by MTT assay. Silencing or over-expression of InnVit gene has little effect on cell migration (as determined by transwell assay) or MMP9 and MMP2 activity.Last, our study further identified the effects of InnVit gene on melanocytes and the relationship of between dilated ER and Tyrosinase by inhibition and over-expression of InnVit gene.By electron microscopicy, obvious swelling of the endoplasmic reticulum (ER) was found in the cells transfected by siRNA for InnVit. Protein levels of Tyrosinase were extraordinarily high following inhibition of InnVit. Further examination revealed Tyrosinase and Calreticulin were co-localized in ER of the transfected cells following siRNA of InnVit, indicating that Tyrosinase could not export from ER effectively, consequently in the abnormity of biological behavior of melanocytes.Collectively, this study provides support that InnVit plays an important role in regulating the proliferation and apoptosis of melanocytes, and revealed the probably mechanisms of functional export of Tyrosinase from ER in vitiligo melanocytes.更多还原