达沙替尼抑制鼻咽癌细胞侵袭及诱导其凋亡的作用

发布时间：2018-01-02 16:03

本文关键词：达沙替尼抑制鼻咽癌细胞侵袭及诱导其凋亡的作用　出处：《安徽医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的鼻咽癌系我国高发的头颈部恶性肿瘤,具有家族及地区聚集性。临床上,就诊的患者大多已为Ⅲ,Ⅳ期,单纯的放射治疗已经不能满足治疗需要,化疗作用有限,需要新的治疗药物出现。鼻咽粘膜的正常组织与癌组织中都有c-Src的表达,癌组织中表达明显增高,且鼻咽癌组织中c-Src表达分别与其与临床分期、预后不良密切相关。故Src也可以作为NPC全身治疗的一个潜在靶点。Src激酶家族系非受体酪氨酸蛋白激酶,其家族成员已发现有9个,这些产物都是具有蛋白酪氨酸激酶活性的蛋白质。它们由特殊结构域组成,分子构型极其相似,参与多条细胞信号转导途径,对细胞的生长,分化,增殖,迁移等生理过程均能发挥重要的作用。其中Src作为人类首先发现的原癌基因c-Src编码的蛋白激酶,其活化与多种人类恶性肿瘤发生及复发转移相关。因此,Src激酶被作为一个治疗癌症的重要靶点,Src激酶抑制剂可能为癌症的治疗提供新的方法。本研究旨在研究Src激酶抑制剂达沙替尼在体外对鼻咽癌细胞的抑制作用。方法选择人鼻咽癌细胞系CNE2,应用不同浓度的达沙替尼处理,分别使用Western blot分析蛋白及磷酸化蛋白表达水平,MTT法绘制细胞生长曲线,AnnexinV/PI双染法检测细胞凋亡及Transwell细胞迁移实验,了解达沙替尼应用后,NPC细胞增殖,凋亡及迁移能力的变化。结果1)应用不同浓度的达沙替尼处理细胞后发现,随着药物浓度的增高,鼻咽癌细胞的增殖能力呈现出被抑制效应明显增强的现象。2)随着药物浓度的增加,phospho-AKT (Ser473)、 phospho-MEK (ser217/221)、phospho-ERK (tyr204)的蛋白水平呈现明显的降低趋势,提示达沙替尼主要抑制MAPK 和 PI3K/AKT信号通路的活性,从而抑制细胞的生长及增殖。3)随着达沙替尼的浓度增加,发现达沙替尼抑制Src的磷酸化活性的能力明显增强,但对Src的总蛋白影响不大。4)达沙替尼能够明显浓度依赖性地诱导细胞发生晚期凋亡,即PI和AnnexinV染色双阳性的细胞比例明显增加。5)达沙替尼在体外能抑制FAK的磷酸化水平,使FAK-SRC复合物的活性显著减弱,从而明显抑制鼻咽癌细胞的迁移能力,且呈浓度依赖性。结论在我们的研究中发现,达沙替尼能通过减少Src激酶磷酸化的水平,使得下游信号转导通路MAPK和PI3K/AKT的活性受到抑制,抑制细胞增殖,诱导凋亡。此外,达沙替尼还能够抑制SRC-FAK复合体的形成,减弱细胞与细胞周围基质之间的黏附作用,从而抑制鼻咽癌细胞的转移。本研究为Src激酶抑制剂Dasatinib将来进一步应用于动物实验,及最终应用于临床治疗晚期鼻咽癌,提供了开端及有力的实验依据。
[Abstract]:Objectivenpc Department of head and neck malignant tumors with high incidence in China, with a family and area aggregation. Clinically, the patients with stage IV have been mostly for III,, radiotherapy alone can not meet the need for treatment, chemotherapy effect, need new drugs. The expression of c-Src in normal tissue and nasopharyngeal carcinoma the mucous membrane of the expression is significantly increased in cancer tissues, and the expression of c-Src and the clinical stages and prognosis are closely related. Therefore, Src NPC can also serve as a systemic treatment of a potential target of.Src kinase family non receptor tyrosine kinase, the family members have been found to have 9 of these products. Is the activity of protein tyrosine kinase protein. They consist of a special domain, molecular conformation is very similar, and participate in multiple cellular signal transduction pathways, cell growth, differentiation, proliferation, migration Shift the physiological process can play an important role. Protein kinase c-Src gene encoding human Src as the first discovered in which the activation of many kinds of human malignant tumor occurrence and metastasis. Therefore, Src kinase is an important target for a cancer treatment, Src kinase inhibitors may provide a new treatment method for cancer. The purpose of this research is to study the Src kinase inhibitor dasatinib in vitro inhibition of nasopharyngeal carcinoma cells. Methods CNE2 cells with different concentrations of dasatinib treatment respectively, using Western analysis of blot protein and phosphorylated protein expression, cell growth curve was drawn by MTT AnnexinV/PI double staining method detection of cell apoptosis and Transwell cell migration experiment, understanding of dasatinib application, NPC cell proliferation, apoptosis and migration. Results 1) with different concentrations Dasatinib treated cells showed that with the increase of drug concentration, the proliferation of nasopharyngeal carcinoma cells showed a significant inhibitory effect was the ability to enhance the phenomenon of.2) with the increase of drug concentration, phospho-AKT (Ser473), phospho-MEK (ser217/221), phospho-ERK (tyr204) protein levels decreased significantly, suggesting that dasatinib mainly the inhibition of MAPK and the activity of the PI3K/AKT signaling pathway, thereby inhibiting the growth and proliferation of.3 cells) with dasatinib concentration increase, ability to find the phosphorylation activity of dasatinib inhibited Src increased significantly, but the total protein has no significant effect on Src.4) Dashti Ni could concentration dependently induced cell induced apoptosis. PI and AnnexinV double staining positive cells significantly increased the proportion of.5 Dashti Ni) can inhibit the phosphorylation of FAK in vitro, the activity of the FAK-SRC complex was significantly reduced Thus, the migration ability of inhibition of nasopharyngeal carcinoma cells, in a dose-dependent manner. Conclusion in our study we found that dasatinib can reduce Src phosphorylation level, the downstream signal transduction pathway of MAPK and the activity of PI3K/AKT was inhibited, inhibit cell proliferation, induce apoptosis. In addition, the formation of dasatinib can also inhibit SRC-FAK complex, weaken the adhesion between the surrounding cells and extracellular matrix, thereby inhibiting the metastasis of nasopharyngeal carcinoma cells. The study of Src kinase inhibitor Dasatinib further applied to animal experiments, and finally applied to the clinical treatment of advanced nasopharyngeal carcinoma, provides the experimental basis for the beginning and strong.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R739.63

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