抗精神病药匹莫齐特抑制人骨肉瘤细胞增殖的作用机制研究

发布时间：2018-04-10 08:42

本文选题：骨肉瘤　切入点：匹莫齐特　出处：《深圳大学》2017年硕士论文

【摘要】：背景:骨肉瘤(Osteosarcoma,OS)是原发性恶性骨肿瘤的一类,在青少年及儿童群体中发生率为2~5/100万人,易产生远端转移,致其患者预后效果较差。研究报道STAT3信号通路的持续激活对骨肉瘤细胞的增殖、存活、药物耐药性等产生直接影响,故而靶向抑制STAT3信号通路的药物将可成为抑制骨肉瘤细胞增殖的有效方法。前期研究工作发现,用于治疗精神病的药物匹莫齐特(Pimozide)能抑制细胞内STAT3信号通路的活化,进而抑制肝癌细胞株MHCC-97L、Hep3B的生长增殖。为此,本研究探讨匹莫齐特抗骨肉瘤的分子机制。目的:研究匹莫齐特对骨肉瘤细胞中STAT3信号通路活化的影响,匹莫齐特对人骨肉瘤细胞是否具有抗癌作用及其相关作用机制。方法:(1)培养人骨肉瘤细胞系;匹莫齐特加药处理骨肉瘤细胞;(2)采用蛋白免疫印迹实验分析STAT3通路有关蛋白p-STAT3的表达水平;通过RT-qPCR检测STAT3通路下游基因的转录表达水平;利用Dual-Luciferase报告基因系统检测STAT3通路的激活程度;(3)通过克隆形成实验,球体形成实验和流式检测骨肉瘤细胞的增殖、凋亡和细胞周期变化;(4)采用活性氧检检测试剂盒测定骨肉瘤细胞内ROS含量;(5)通过细胞增殖实验观察过表达CAT基因后对匹莫齐特效用的影响;(6)开展染色质免疫共沉淀实验分析CAT基因与STAT3的关系。结果:(1)匹莫齐特可以降低骨肉瘤细胞内STAT3信号活性,主要表现为降低信号激活标志物p-STAT3表达水平、下调STAT3信号下游基因MYC、BCL-xl和MCL-1的转录表达水平,并且抑制STAT3-Luc报告基因的活性;(2)匹莫齐特抑制骨肉瘤细胞系U2OS、MG-63与SW1353细胞的增殖和骨肉瘤细胞的克隆形成和球体形成能力,表明匹莫齐特能抑制骨肉瘤细胞的干性能力,主要表现为下调干性相关因子Sox2、Nanog和c-Myc蛋白表达、以及增强细胞对5-FU诱导增殖抑制效果和降低细胞内侧群细胞(Side population,SP)的比例;(3)匹莫齐特可诱导骨肉瘤细胞发生凋亡,并使细胞的G0/G1期产生阻滞;(4)匹莫齐特能抑制细胞Erk信号激活影响骨肉瘤细胞的增殖活性,表现为下调p-Erk蛋白的表达;Erk通路抑制剂无法增强匹莫齐特对细胞的增殖抑制效果;(5)匹莫齐特抑制细胞内CAT基因表达而促进ROS产生,但对SOD1、SOD2和GPX1等基因表达没有影响;CAT过表达后能减弱匹莫齐特对骨肉瘤细胞的增殖抑制作用;(6)生物信息学推测CAT基因的调控序列中可能含有两个STAT3结合的潜在位点;ChIP实验结果显示STAT3蛋白可结合在CAT基因的启动子区。结论:本课题研究发现匹莫齐特能有效地抑制骨肉瘤细胞内STAT3信号活化,对骨肉瘤细胞具有抗癌作用;并提出匹莫齐特的抗癌分子机制是通过抑制骨肉瘤细胞中STAT3信号活性而降低CAT基因的表达,进而产生大量ROS导致细胞产生增殖抑制和细胞凋亡。匹莫齐特可作为STAT3的潜在靶向抑制剂,有望成为治疗骨肉瘤的潜在药物或者先导化合物,为临床应用奠定了基础。
[Abstract]:Background: Osteosarcoma OS (Osteosarcoma) is a kind of primary malignant bone tumor. The incidence rate of Osteosarcoma Osteosarcoma is 2 to 5 / 1 million in adolescents and children.It is reported that the continuous activation of STAT3 signaling pathway has a direct effect on the proliferation, survival and drug resistance of osteosarcoma cells. Therefore, drugs targeted at inhibiting STAT3 signaling pathway will become an effective method to inhibit the proliferation of osteosarcoma cells.Previous studies have shown that pimozide-pimozideinhibits the activation of intracellular STAT3 signaling pathway and thus inhibits the growth and proliferation of hepatocellular carcinoma cell line MHCC-97LnHep3B.Therefore, the molecular mechanism of pimozide against osteosarcoma was investigated.Aim: to study the effect of pimozide on the activation of STAT3 signaling pathway in osteosarcoma cells.Methods the human osteosarcoma cell line was cultured with 1) and the osteosarcoma cell line was treated with pimochizide. The expression level of protein p-STAT3 related to the STAT3 pathway was analyzed by Western blot assay, and the transcription expression level of the downstream gene of the STAT3 pathway was detected by RT-qPCR.The activation of STAT3 pathway was detected by Dual-Luciferase reporter gene system. The proliferation of osteosarcoma cells was detected by clone formation assay, globular formation assay and flow cytometry.Apoptosis and change of cell cycle: we used reactive oxygen species assay kit to detect ROS content in osteosarcoma cells. We observed the effect of CAT gene expression on the specific effect of pimozil by cell proliferation assay.The relationship between CAT gene and STAT3 was analyzed.Results Pimozide could decrease the activity of STAT3 signal in osteosarcoma cells, mainly by decreasing the expression level of signal activation marker p-STAT3 and down-regulating the transcription expression of MYCnBCL-xl and MCL-1 in the downstream genes of STAT3 signal.The inhibitory effect of pimozide on the proliferation of osteosarcoma cell line U2OS-1 MG-63 and on the ability of osteosarcoma cell line to clone and globulate osteosarcoma cell line U2OS- MG-63 and osteosarcoma cell line was also inhibited by pimozide, which indicated that pimozide could inhibit the dry ability of osteosarcoma cells.The main findings were as follows: down-regulating the expression of dry-related factor Sox2Nanog and c-Myc, and enhancing the inhibitory effect of cells on 5-FU induced proliferation and decreasing the proportion of cell medial group cell side population SPs. The pimozide could induce apoptosis in osteosarcoma cells, and the apoptosis of osteosarcoma cells could be induced by pimozide.Pimozil could inhibit the activation of Erk signal and affect the proliferation of osteosarcoma cells.The results showed that the down-regulation of the expression of p-Erk protein and Erk pathway inhibitor could not enhance the inhibitory effect of pimozide on cell proliferation. Pimozide inhibited the expression of CAT gene and promoted the production of ROS.However, the expression of SOD1, SOD2 and GPX1 genes did not affect the overexpression of catalase, which could attenuate the inhibitory effect of pimozide on the proliferation of osteosarcoma cells.) Bioinformatics suggested that the regulatory sequence of CAT gene might contain two potential STAT3 binding sites.The results of ChIP showed that STAT3 protein could bind to the promoter region of CAT gene.Conclusion: in this study, we found that pimozide can effectively inhibit the activation of STAT3 signal in osteosarcoma cells and has anticancer effect on osteosarcoma cells.It is suggested that the molecular mechanism of pimozide is to reduce the expression of CAT gene by inhibiting the activity of STAT3 signal in osteosarcoma cells, which leads to the proliferation inhibition and apoptosis of osteosarcoma cells due to the production of a large number of ROS.As a potential target inhibitor of STAT3, pimozide is expected to be a potential drug or lead compound for the treatment of osteosarcoma, which lays the foundation for clinical application.
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R738

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