PHB2表达沉默对横纹肌肉瘤细胞肿瘤生物学行为的影响及作用机制的初步研究

发布时间：2018-08-02 14:30

【摘要】：PHB2(Prohibitin2,37KD)是进化上高度保守的多功能蛋白,广泛分布于细菌、酵母、原虫、植物、果蝇以及哺乳类等多种生物细胞中,参与细胞的增殖、分化、迁移、凋亡及线粒体形态发生等生理过程,以及调节细胞周期、基因转录、和细胞内信号转导。然而,对PHB2的研究仍处于初级阶段,很多功能及机制仍不清晰。横纹肌肉瘤(Rhabdomyosarcoma)是源于向横纹肌分化的原始间叶细胞,并由不同分化程度的横纹肌细胞组成的软组织恶性肿瘤。临床表现的多样性、病理改变的多重性以及发病部位的多变性,使横纹肌肉瘤成为最复杂的小儿肿瘤之一。我们前期的研究发现PHB2能抑制骨骼肌的分化,而横纹肌肉瘤正是以骨骼肌祖细胞过度增殖及分化缺陷为主要特征,这些证据提示PHB2与横纹肌肉瘤形成可能具有一定的相关性。故本文采用RNA干涉技术,使RD细胞系(Rhabdomyosarcoma cells)中的PHB2表达沉默,观察PHB2表达沉默对RD细胞增殖、分化、迁移等方面的影响并对相关作用机制进行初步探究,为进一步探究PHB2在横纹肌肉瘤发生发展过程中的生物学作用提供实验基础和理论依据。主要研究内容如下:1、通过实时阻抗细胞分析技术检测到PHB2表达沉默后,RD细胞活力降低。流式细胞术的结果显示,PHB2表达沉默后细胞G1期的比例增加,细胞被阻滞在G1/S期,从而抑制了 RD细胞的增殖;2、软琼脂中克隆形成实验显示PHB2表达沉默后,RD细胞的锚定非依赖性克隆形成能力下降,说明RD细胞的转化特性减弱;3、在RD细胞中,部分PHB2疑似特异地定位于细胞核的核仁区域,而且RT-PCR方法初步证明了 PHB2表达沉默后,核仁中合成的45SrRNA含量下调,提示PHB2可能参与核仁功能的调控,这可能是其参与调控RD细胞增殖过程的机制之一;4、PHB2表达沉默后,RD细胞中肌细胞分化前期标志物myogenin的表达量上调,说明PHB2表达沉默能增强RD细胞的分化能力。通过荧光素酶报告基因活性检测进一步证明PHB2表达沉默后myogenin的启动子活性增加,提示了PHB2与细胞分化相关的一种可能的机制;5、采用Transwell检测细胞迁移发现PHB2表达沉默对RD细胞的迁移能力没有影响。
[Abstract]:PHB2 (inhibitor 2N 37KD) is an evolutionarily conserved multifunctional protein, widely distributed in bacteria, yeast, protozoa, plants, Drosophila, mammalian and other biological cells, participating in cell proliferation, differentiation and migration. Apoptosis, mitochondrial morphogenesis and other physiological processes, as well as regulation of cell cycle, gene transcription, and intracellular signal transduction. However, the study of PHB2 is still in its infancy, and many functions and mechanisms are still unclear. Rhabdomyosarcoma (Rhabdomyosarcoma) is a kind of soft tissue malignant tumor derived from primitive mesenchymal cells differentiated into rhabdomyosarcoma and composed of striated muscle cells of different degrees. The multiplicity of clinical manifestations, the multiplicity of pathological changes and the variability of the site of the disease make rhabdomyosarcoma one of the most complicated tumors in children. Our previous studies have found that PHB2 can inhibit skeletal muscle differentiation and rhabdomyosarcoma is characterized by excessive proliferation and differentiation defect of skeletal muscle progenitor cells. These results suggest that PHB2 may be related to the formation of rhabdomyosarcoma. Therefore, the expression of PHB2 in Rd cell line (Rhabdomyosarcoma cells) was silenced by RNA interference technique. The effects of PHB2 expression silencing on proliferation, differentiation and migration of Rd cell line were observed. To further explore the biological role of PHB2 in the development of rhabdomyosarcoma provides experimental and theoretical basis. The main contents are as follows: 1. The activity of Rd cells decreased after PHB2 expression silencing was detected by real time impedance cell analysis. The results of flow cytometry showed that the proportion of cells in G1 phase was increased after the expression of PHB2 was silenced, and the cells were blocked in G1 / S phase. Thus, the proliferation of Rd cells was inhibited. Clone formation assay in soft Agar showed that the ability of anchoring independent clone formation of Rd cells decreased after PHB2 expression silencing, which indicated that the transformation characteristics of Rd cells were weakened in R D cells. Some PHB2 were suspected to be located specifically in the nucleolus region of the nucleus, and the RT-PCR method preliminarily proved that the 45SrRNA content in the nucleolus was down-regulated after the silencing of PHB2 expression, suggesting that PHB2 might be involved in the regulation of nucleolar function. This may be one of the mechanisms involved in regulating the proliferation of Rd cells. After the silencing of PHB2 expression, the expression of myogenin in Rd cells is up-regulated, indicating that the silencing of PHB2 expression can enhance the differentiation ability of Rd cells. The detection of luciferase reporter gene activity further proved that the promoter activity of myogenin increased after PHB2 expression silencing. It is suggested that PHB2 may be a possible mechanism related to cell differentiation. The expression silencing of PHB2 has no effect on the migration ability of Rd cells by using Transwell to detect cell migration.
【学位授予单位】：东北师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R738.7

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