胰腺癌细胞抵抗吉西他滨诱导的细胞死亡的机制研究

发布时间：2018-04-20 08:10

本文选题：胰腺癌 + 化疗耐药　；参考：《第四军医大学》2015年硕士论文

【摘要】：胰腺癌是死亡率最高的恶性肿瘤之一,胰腺癌的致死性主要是由于其化疗药物抵抗能力,这与胰腺癌细胞死亡信号通路的改变密切相关。以往大家普遍认为肿瘤细胞化疗耐药的机制一般为对细胞凋亡的抵抗,细胞凋亡的抵抗机制一直是研究的热门话题。然而越来越多的证据表明,肿瘤细胞死亡方式呈现多样化。深入理解化疗药物诱导的肿瘤细胞死亡形式及信号通路的变化对于肿瘤化疗耐药的克服及药靶的开发具有非常重要的现实意义。程序性坏死是近几年发现的另一种程序性细胞死亡形式,与凋亡相似,是可调控的细胞死亡形式。一般在凋亡信号通路受到阻断,caspase活性受到抑制的条件下发生,这一特质也恰恰使程序性坏死具有了克服肿瘤化疗耐药的潜质,而且使得程序性坏死在耐药方面的研究成为热点。但是,程序性坏死在传统化疗药物诱导的肿瘤细胞死亡抵抗中是否发挥作用,凋亡和程序性坏死在肿瘤细胞死亡抵抗中发挥怎样的作用,调控肿瘤细胞死亡形式及死亡抵抗的关键分子有哪些还不明确,本研究就是要针对这些问题进行初步探究。在本研究中,我们首先分析了168例胰腺癌病人样本,发现RIP3的表达水平与胰腺癌病人的总体生存时间显著相关,RIP3高表达是胰腺癌病人死亡的危险因素。然后我们在细胞水平进行了研究,我们筛选出能够发生程序性坏死的胰腺癌细胞株CFPAC-1,我们发现吉西他滨诱导该细胞的死亡不会被全caspase抑制剂Z-VAD-fmk阻断,却能够被程序性坏死抑制剂Nec-1部分阻断,且透射电镜结果显示,吉西他滨能够诱导CFPAC-1发生凋亡和坏死,。我们用吉西他滨诱导CFPAC-1细胞获得了IC50为20μM的耐药细胞株R1、R2。另外,我们发现该耐药细胞株获得了抵抗凋亡和程序性坏死的能力。我们首次在肿瘤耐药模型中研究了程序性坏死的作用。我们发现在胰腺癌耐药细胞株中,RIP3、CD147、p-Drp1的表达水平显著上调。干涉RIP3能够促进吉西他滨诱导的耐药肿瘤细胞的死亡,对CFPAC-1细胞没有显著作用。另外,干涉CD147或者利用CD147的单克隆抗体Hab18/6H8阻断联合吉西他滨均能逆转CFPAC-1(R)吉西他滨耐药。我们还发现Drp-1 S637位去磷酸化及线粒体分裂参与了吉西他滨诱导的胰腺癌细胞死亡及耐药。最后,我们发现CD147能够促进Drp1磷酸化,抑制Drp1二聚抑制线粒体分裂,从而抑制吉西他滨诱导的细胞死亡。RIP3能够通过调控CD147及其下游信号分子Bcl-2、Drp-1、NF-kB等参与肿瘤细胞死亡抵抗。
[Abstract]:Pancreatic cancer is one of the most fatal malignant tumors, and its mortality is mainly due to its resistance to chemotherapy drugs, which is closely related to the change of death signal pathway in pancreatic cancer cells. In the past, it was generally believed that the mechanism of chemotherapeutic resistance of tumor cells is the resistance to apoptosis, and the mechanism of resistance to apoptosis has always been a hot topic. However, there is increasing evidence that tumor cells die in a variety of ways. It is very important to understand the changes of tumor cell death and signal pathway induced by chemotherapeutic drugs for overcoming chemotherapeutic resistance and developing drug targets. Programmed necrosis is another kind of programmed cell death found in recent years, which is similar to apoptosis and can be regulated by cell death. The activity of caspase occurs when the apoptotic signaling pathway is blocked, which makes the programmed necrosis have the potential to overcome the chemotherapeutic resistance of tumor, and makes the research on the drug resistance of the programmed necrosis become a hot topic. However, whether programmed necrosis plays a role in tumor cell death resistance induced by traditional chemotherapeutic drugs, and what role apoptosis and programmed necrosis play in tumor cell death resistance, It is not clear which key molecules are involved in the regulation of tumor cell death forms and death resistance. In this study, we first analyzed 168 patients with pancreatic cancer. We found that the expression of RIP3 was significantly correlated with the overall survival time of patients with pancreatic cancer, and the high expression of RIP3 was a risk factor for death in patients with pancreatic cancer. Then we studied it at the cell level, and we screened the pancreatic cancer cell line CFPAC-1, which could cause programmed necrosis, and we found that gemcitabine-induced cell death was not blocked by the whole caspase inhibitor Z-VAD-fmk. The results of transmission electron microscope showed that gemcitabine could induce apoptosis and necrosis of CFPAC-1. CFPAC-1 cells were induced by gemcitabine to obtain a 20 渭 M IC50 resistant cell line R _ (1) and R _ (2). In addition, we found that the drug resistant cell line acquired the ability to resist apoptosis and programmed necrosis. For the first time, we studied the role of programmed necrosis in tumor resistance models. We found that the expression of RIP3, CD147, p-Drp1 was significantly up-regulated in drug-resistant pancreatic cancer cell lines. Intervention of RIP3 could promote the death of drug resistant tumor cells induced by gemcitabine, but had no significant effect on CFPAC-1 cells. In addition, interfering with CD147 or blocking the combination of gemcitabine with Hab18/6H8, a monoclonal antibody of CD147, could reverse the resistance to gemcitabine. We also found that Drp-1 S637 dephosphorylation and mitochondrial division were involved in gemcitabine-induced pancreatic cancer cell death and drug resistance. Finally, we found that CD147 can promote Drp1 phosphorylation, inhibit Drp1 dimerization, inhibit mitochondrial division, and thus inhibit gemcitabine-induced cell death. RIP3 can participate in tumor cell death resistance by regulating CD147 and its downstream signaling molecule Bcl-2NF-kB.
【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】：R735.9

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