SDF-1-CXCR4与HIF-1在卵巢癌细胞转移中相互作用及机制研究

发布时间：2018-07-14 20:08

【摘要】：研究背景卵巢癌系女性生殖系统恶性肿瘤之一,其死亡率位高不下,较高的侵袭性和转移性是导致患者以后不良的主要因素。大约75%左右患者一经诊断已为晚期,由于患者已发生远处转移,致使治疗棘手,患者已失去最佳治疗时机预后极差。因此,了解卵巢癌的转移机制对临床治疗及提高患者预后有着十分重要的作用。肿瘤的转移是一个复杂的多因素过程,趋化因子则在肿瘤转移过程中起着至关重要的作用。基质细胞衍生因子SDF-1(stromal derived factor-1,又称CXCR12)作为趋化因子家族中的一员,已成为近些年来研究的热点和重点。SDF-1主要与其相应的受体CXCR4结合从而诱导卵巢癌的转移进程,这一过程主要由于激活了下游的p38MAPK/PI-3K信号通路。p38MAPK/PI-3K信号通路被公认为是肿瘤转移过程中一条经典的信号通路,缺氧诱导因子-1(hypoxia inducible factor-1,HIF-1)发挥其作用被报道也依靠其上游此通路的激活作用。鉴于两者发挥作用依赖共同的信号通路,我们试想两者是否具有相关性,其相关机制是否为p38MAPK/PI-3K信号通路作用?因此我们的实验主要研究SDF-1-CXCR4与HIF-1在卵巢癌细胞转移中相互作用及机制,为卵巢癌的临床治疗提供新的思路。本实验主要研究SDF-1α与HIF-1α在卵巢癌中的相关作用及其机制,首次将两者在卵巢癌中联系起来,本研究证明了两者在卵巢癌组织中呈正相关,并对其具体通路进行了研究,阐明了两者间作用机制。研究目的1.SDF-1α与HIF-1α在卵巢癌组织中的表达是否相关。2.SDF-1-CXCR4生物轴是否会激活HIF-1的表达。3.HIF-1转录和表达的增加是否会促进CXCR4的表达。4.SDF-1对PI3K/AKT、MAPK信号通路和HIF-1激活作用是否一致5.探讨HIF-1是否会进一步促进CXCR4的表达以及VEGF的表达从而促进细胞的迁移。研究方法1.应用免疫组织化学法检测卵巢癌组织中SDF-α1与HIF-1α的表达;2.实时定量RT-PCR检测各个细胞组中HIF-1α的m RNA水平。3.运用Western-blot检测细胞中AKT/p-AKT、p-38/p-p38、HIF-1α、CXCR4的表达水平。4.采用ELISA检测VEGF的含量。5.Tranwell侵袭小室、划痕实验检测各组细胞的迁移。研究结果1.SDF-1α与HIF-1α在卵巢癌组织中的表达呈正相关(r=0.475,P0.01);2.q RT-PCR结果证明,随着加入外源性SDF-1量及时间增加,HIF-1α的m RNA水平依次增加(P0.05)。3.Western-blot分析显示,control、control+SDF-1、control+CXCR4封闭抗体三组中,control+SDF-1组中p-AKT、p-p38量明显增高(P0.01);control、control+SDF-1、control+CXCR4封闭抗体、control+SDF-1α+LY294002四组中,control+SDF-1组中HIF-1水平明显增高(P0.01);control+SDF-1、卵巢癌细胞转染HIF sh RNA+SDF-1两组中,control+SDF-1组CXCR4量明显增高(P0.01)。4.ELISA检测结果证明,control+SDF-1、卵巢癌细胞转染HIF sh RNA+SDF-1两组中,control+SDF-1组VEGF量明显增高(P0.05)。5.Tranwell侵袭实验结果:SDF-1组较空白组和对照组HIF sh RNA+SDF-1明显增强,有显著性差异(P0.01)。6.划痕实验结果:对于12h、24h、36h后划痕处的距离进行分析SDF-1组较空白组和对照组HIF sh RNA+SDF-1明显增强,有显著性差异(P0.05)。结论1.上皮性卵巢癌组织中SDF-1α和HIF-1α呈正相关。2.SDF-1-CRCX4生物轴促进HIF-1α表达。3.SDF-1促进PI3K/AKT、MAPK信号通路和HIF-1激活。4.HIF-1促进CXC4及VEGF的表达从而促进细胞的迁移。
[Abstract]:The research background of ovarian cancer is one of the malignant tumors of female reproductive system. The mortality rate is high, the higher invasion and metastasis are the main factors that cause the patient's bad. About 75% of the patients have been diagnosed as late, because the patients have had distant metastasis, which makes the treatment difficult and the patient has lost the best treatment time. Therefore, it is very important to understand the mechanism of ovarian cancer metastasis to the clinical treatment and to improve the prognosis of the patients. Metastasis is a complex multifactor process, and chemokine plays a vital role in the process of tumor metastasis. The matrix cell derived factor SDF-1 (stromal derived factor-1, also known as CXCR12) A member of the chemokine family has become a hot spot in recent years and the focus of.SDF-1 is mainly associated with its corresponding receptor CXCR4 to induce ovarian cancer metastasis. This process is mainly due to the activation of the downstream p38MAPK/PI-3K signaling pathway.P38MAPK/PI-3K signal is recognized as a classic in the process of tumor metastasis. The role of -1 (hypoxia inducible factor-1, HIF-1) is reported to depend on the activation of the upstream pathway. In view of the dependence of the two on the common signal pathway, we try to think whether the two are related, and whether the phase switching system is a p38MAPK/PI-3K signaling pathway? The experiments mainly study the interaction and mechanism of SDF-1-CXCR4 and HIF-1 in ovarian cancer cell metastasis and provide new ideas for the clinical treatment of ovarian cancer. This experiment mainly studies the relationship between SDF-1 alpha and HIF-1 alpha in ovarian cancer and its mechanism. The two are first linked in ovarian cancer. This study proves that both of them are in the ovarian cancer group. There is a positive correlation in the fabric, and the specific pathways are studied and the mechanism of interaction between the two is clarified. Whether or not the expression of 1.SDF-1 alpha and HIF-1 a in ovarian cancer tissue is related to whether the.2.SDF-1-CXCR4 biological axis activates HIF-1 expression and the increase of.3.HIF-1 transcription and expression will promote the expression of CXCR4 in.4.SDF-1 to PI3K/AKT, MAPK Whether the signal pathway and HIF-1 activation are consistent 5. to explore whether HIF-1 can further promote the expression of CXCR4 and the expression of VEGF to promote cell migration. Method 1. the expression of SDF- alpha 1 and HIF-1 alpha in ovarian cancer tissues was detected by immunohistochemistry; 2. real-time quantitative RT-PCR was used to detect the m RNA level of HIF-1 alpha in each cell group. .3. used Western-blot to detect the expression level of AKT/p-AKT, p-38/p-p38, HIF-1, and CXCR4 in the cell.4. using ELISA to detect the VEGF content.5.Tranwell invasion of the small chamber and the scratch test to detect the migration of the cells. Adding exogenous SDF-1 and increasing the time, the m RNA level of HIF-1 alpha increased in turn (P0.05).3.Western-blot analysis showed that control, control+SDF-1, control+CXCR4 closed antibody three groups, control+SDF-1 group p-AKT, p-p38 volume increased significantly. In the group control+SDF-1, the level of HIF-1 increased significantly (P0.01), and control+SDF-1, in the HIF sh RNA+SDF-1 two group transfected with ovarian cancer cells, the CXCR4 content of control+SDF-1 group increased significantly (P0.01).4.ELISA detection results. The results of the invasion experiment: the HIF sh RNA+SDF-1 in the SDF-1 group was significantly higher than that in the blank group and the control group. There was a significant difference (P0.01).6. scratch test results: the distance between the 12h, 24h, and the scratch marks after 36h was analyzed. The SDF-1 group was significantly enhanced in the SDF-1 group than the blank group and the control group, and there were significant differences. Conclusion 1. epithelial ovarian cancer tissues were found. The positive correlation of SDF-1 alpha and HIF-1 alpha to.2.SDF-1-CRCX4 biological axis promotes the expression of HIF-1 a.3.SDF-1 to promote PI3K/AKT, MAPK signaling pathway and HIF-1 activate.4.HIF-1 to promote the expression of CXC4 and VEGF, thus promoting cell migration.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.31

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