OCT4调控STAT3对细胞色素C活性影响及其研究

发布时间：2018-07-03 14:21

本文选题：OCT4 + STAT3　；参考：《山东大学》2017年硕士论文

【摘要】：背景与目的OCT4是位于细胞核内的POU家族关键转录因子之一,胚胎于细胞、生殖干细胞中常见到OCT4的大量表达.OCT4对于ESC的维干及ESC的自我更新意义重大。最新的研究发现,OCT4在胚胎性癌、精原细胞瘤以及生殖系统以外的某些恶性肿瘤中也有着高表达,意味着OCT4在维持肿瘤干细胞的分化潜能中也有作用,当细胞中OCT4维持特定浓度时,细胞保持其干性,当细胞中OCT4表达下调时,细胞分化成具有各种功能的细胞,丧失其干性。STAT3是STATs家族中的重要一员,STAT3的功能是参与细胞的增殖、分化、凋亡,研究表明,持续激活的STAT3可引发癌细胞的恶化和扩散。OCT4、STAT3影响干细胞的分化及癌细胞EMT作用的机制并不完善,本文通过研究OCT4抑制STAT3进线粒体,使细胞色素C(cytochrome C)活性降低的过程,在能量代谢角度探究OCT4在干细胞的分化及癌细胞EMT作用的机制。方法用PCR技术,转化技术制作STAT3、OCT4质粒,OCT4阶段体、点突变体,以人肾上皮293T细胞,宫颈癌细胞系HeLa为材料,转入外源STAT3、OCT4、及其突变体,确定OCT4与STAT3在细胞质存在相互作用关系,并确定OCT4与STAT3结合的关键位点。用线粒体分离术,免疫荧光蛋白[质]印迹法(Westem blot)等方法,测定转入OCT4质粒后STAT3入线粒体的量及细胞色素C活性,测定细胞ATP的生成量。结果在人肾上皮293T细胞中转入外源OCT4后STAT3入线粒体量明显减少,细胞呼吸作用降低,使用试剂盒测得细胞ATP产生量减少。在人肾上皮293T细胞,颈癌细胞系HeLa分别转入外源OCT4全长、OCT4截断体、OCT4点突变体后,提取细胞线粒体蛋通过白免疫荧光蛋白[质]印迹法测得OCT4 144位影响OCT4与STAT3的结合,通过试剂盒测得,转入OCT4的癌细胞细胞色素C活性降低。ATP量减少。结论OCT4可以通过抑制STAT3进线粒体,使得STAT3与细胞色素C在线粒体内的的结合变少,进而使细胞的ATP合成量减少。STAT3与OCT4的结合与OCT4第144位点相关。推测在普通细胞转化为IPS细胞时,癌细胞发生EMT作用转变为具有干性癌细胞时,OCT4均通过STAT3通路调控细胞色素C,降低细胞呼吸链活性从而调控细胞的干性及分化。
[Abstract]:Background & objective OCT4 is one of the key transcription factors of POU family located in the nucleus. The high expression of OCT4 is often seen in embryonic cells and reproductive stem cells. OCT4 is of great significance for the maintenance of ESC and the self-renewal of ESC. New research has found that OCT4 is also highly expressed in embryonic carcinomas, seminoma, and some malignancies outside the reproductive system, suggesting that OCT4 also plays a role in maintaining the differentiation potential of tumor stem cells. When OCT4 is maintained at a specific concentration, the cell remains dry. When the expression of OCT4 is down-regulated, the cells differentiate into cells with various functions. The loss of their dryness. STAT3 is an important member of the stats family. The function of STAT3 is involved in cell proliferation. Differentiation, apoptosis, studies have shown that sustained activation of STAT3 can cause the deterioration and diffusion of cancer cells. OCT4STAT3 affects the differentiation of stem cells and the mechanism of EMT of cancer cells is not perfect. In this paper, we studied the inhibition of STAT3 into mitochondria by OCT4. In the process of decreasing cytochrome C (cytochrome C) activity, the mechanism of OCT4 in differentiation of stem cells and the role of cancer cells in EMTs was explored in the view of energy metabolism. Methods STAT3OCT4 plasmids and point mutants were prepared by PCR and transformation techniques. Human renal epithelial 293T cells and cervical cancer cell line HeLa were transformed into exogenous STAT3OCT4 and its mutants. The interaction between OCT4 and STAT3 in cytoplasm was determined. The key sites for the binding of OCT4 to STAT3 were identified. The amount of STAT3 into mitochondria and cytochrome C activity were measured by mitochondrial isolation and Western blot. Results after transfection of exogenous OCT4 into 293T cells of human renal epithelium, the amount of STAT3 into mitochondria decreased, the cell respiration decreased, and the ATP production of cells was decreased by using kit. After human renal epithelial 293T cells and cervical cancer cell line HeLa were transferred into exogenous OCT4 full-length OCT4 truncated OCT4 point mutants, the mitochondrial eggs were extracted. The binding of OCT4 to STAT3 was determined by immunofluorescence protein [qualitative] blotting. The activity of pigment C in OCT4 cells was decreased. ATP content was decreased. Conclusion by inhibiting STAT3 into mitochondria, OCT4 can reduce the binding of STAT3 to cytochrome C in mitochondria, and then decrease the amount of ATP synthesis. The binding of STAT3 to OCT4 is related to the 144 site of OCT4. It is speculated that when the normal cells are transformed into IPS cells, the EMT action of cancer cells changes to that of dry cancer cells. Both of them regulate cytochrome C through STAT3 pathway, decrease the activity of cell respiratory chain and regulate the dryness and differentiation of the cells.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q25

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