中华皮肤科杂志 ›› 2010, Vol. 43 ›› Issue (4): 269-272.

• 论著 • 上一篇    下一篇

InnVit基因对B10BR细胞增殖、凋亡及细胞周期的影响

林福全1,关翠萍1,刘东银1,洪为松1,周妙妮2,许爱娥3   

  1. 1. 杭州市第三人民医院
    2. 杭州市第三人民医院皮肤科
    3. 安徽医科大学附属杭州市第三人民医院皮肤科
  • 收稿日期:2009-07-01 修回日期:2009-07-31 出版日期:2010-04-15 发布日期:2010-04-07
  • 通讯作者: 林福全 E-mail:leo885528@gmail.com
  • 基金资助:

    InnVit基因调控黑素细胞生物学功能及其分子机制;InnVit基因在白癜风发生中的作用研究;CpG-ODN经Akt/mTOR抗UVB诱导的角质形成细胞凋亡的保护机制研究

Effect of InnVit gene expression on proliferation, apoptosis and cell cycle of mouse melanocyte cell line B10BR

  • Received:2009-07-01 Revised:2009-07-31 Online:2010-04-15 Published:2010-04-07

摘要:

目的 分析InnVit基因的抑制和过表达对永生化黑素细胞株B10BR细胞增殖、周期及凋亡的影响,探讨InnVit基因的生物学功能及其对白癜风中黑素细胞缺失的影响。方法 化学合成InnVit基因特异性siRNA片段以及构建过表达质粒载体P3XF-P120,lipofectamineTM 2000脂质体方法转染B10BR细胞。半定量RT-PCR法检测InnVit基因mRNA水平;Western印迹检测该蛋白水平变化;MTT法测定细胞增殖情况;流式细胞技术分析细胞周期和凋亡的变化。结果 InnVit基因抑制后mRNA和蛋白表达水平明显低于对照组,InnVit基因过表达质粒组mRNA和蛋白表达水平显著高于对照组;转染后24、48、72 h抑制组的细胞存活量极显著下降(P < 0.01),过表达质粒组的细胞存活量在48和72 h极显著增加(P < 0.01)。转染后48 h基因抑制组的早期凋亡率从(10.24 ± 1.00)%升至(37.34 ± 3.26)%,过表达质粒组早期凋亡率从(14.58 ± 1.49)%降至(8.43 ± 0.86)%,与对照组比较,差异均有统计学意义(P < 0.01);转染48 h基因抑制组的G1期细胞由(56.11 ± 5.46)%增至(69.76 ± 6.08)%(P < 0.05),过表达质粒组的G1期细胞由(55.14 ± 5.65 )%降至(29.33 ± 3.01)%(P < 0.01)。结论 InnVit基因抑制使细胞增殖能力下降,促进细胞凋亡;InnVit基因的过表达增强了细胞的增殖能力,抑制细胞凋亡。其增殖能力的改变可能是通过细胞周期的调控调节的。

关键词: InnVit基因, B10BR, 白癜风, 细胞增殖, 细胞凋亡, 细胞周期

Abstract:

Objective To assess the effect of InnVit gene expression on cell proliferation, apoptosis and cell cycle of B10BR cells, and to explore the biological function of InnVit gene and its influence on the loss of melanocytes in vitiligo. Methods InnVit gene-targeting siRNA was synthesized, and overexpression plasmid P3XF-P120 was constructed. B10BR cells were classified into 4 groups to be transfected with control siRNA (negative control), siRNA targeting InnVit gene (siRNA group), empty plasmid (plasmid control group), and P3XF-P120 plasmid (P3XF-P120 group), respectively. After additional culture for various periods, the mRNA and protein levels of InnVit gene were detected by RT-PCR and Western blot, respectively, cell surviva1 by MTT assay, cell cycle and apoptosis by flow cytometry. Results The expression of InnVit mRNA and protein significantly increased in P3XF-P120 group compared with the plasmid control group, but decreased in siRNA group compared with the negative control group (all P < 0.01). After the transfection, the survival rate of cells was significantly lower in siRNA group than in negative control group at 24, 48 and 72 hours, but higher in P3XF-P120 group than in plasmid control group at 48 and 72 hours (all P < 0.01). At 48 hours after the transfection, the early apoptosis rate and proportion of cells in G1 phase increased from (10.24 ± 1.00)% and (56.11 ± 5.46)% in negative control group to (37.34 ± 3.26)% and (69.76 ± 6.08)%, in siRNA group, respectively (P < 0.01 or 0.05), from (14.58 ± 1.49)% and (55.14 ± 5.65)% in plasmid control group to (8.43 ± 0.86)% and (29.33 ± 3.01)%, in P3XF-P120 group, respectively (both P < 0.01). Conclusions The suppression of InnVit gene efficiently inhibits the proliferation and induces apoptosis of B10BR cells, and vice versa. Therefore, the regulation of cell cycle may affect the proliferation of B10BR cells.

Key words: InnVit gene, B10BR, vitiligo, proliferation, cycle, apoptosis