地喹氯铵对神经胶质瘤的作用及其机制研究

发布时间：2018-04-09 00:39

本文选题：神经胶质瘤　切入点：地喹氯铵　出处：《吉林大学》2014年博士论文

【摘要】：神经胶质瘤是最常见的颅内肿瘤，其特点是病程短、治愈难、病死率高。目前神经胶质瘤的治疗方式主要包括了手术切除、放射治疗、化学治疗、免疫治疗、分子靶向治疗等。手术治疗为该病治疗首选方法，但由于神经胶质瘤自身特点，肿瘤组织与正常脑组织界限模糊、不易辨别，且肿瘤可生长延伸至多个重要功能区域，使得手术治疗彻底切除病灶难度很大。单纯性手术治疗往往很难达到使肿瘤细胞数量减少到机体免疫系统能消灭肿瘤细胞的程度，因此手术治疗效果往往不佳，手术后的MRI复查显示脑胶质瘤患者中仅60%左右的可以达到影像学全切除。所以目前神经胶质瘤的治疗方式是以手术切除为基础，结合放射治疗、化学治疗、免疫治疗、分子靶向治疗等方法形成的综合治疗策略。但胶质瘤治疗仍缺乏突破性进展。探索胶质瘤创新诊疗技术，一直是神经外科学的研究热点。烷化剂替莫唑胺（temozolomide，TMZ）在临床上的应用，使恶性脑胶质瘤患者化疗取得了重大进展，人们重新开始重视化疗药物的抗胶质瘤作用。但尽管TMZ的应用使得神经胶质瘤的治疗效果有了显著的改善，但因其难以通过血脑屏障、肿瘤及周边组织间隙静水压高等导致的药物有效浓度降低，长期用药引起肿瘤细胞产生的耐药性，对机体的不良反应，分子遗传学改变导致的治疗困难等，均会对神经胶质瘤治疗产生不利影响。因此，胶质瘤的治疗仍面临许多困难。寻找具有低毒副作用、能够靶向作用于肿瘤的药物，一直是抗肿瘤药物研发的目标。地喹氯铵（dequalinium chloride，DECA）是人工合成的喹啉衍生物，含有两个孤立的喹啉芳杂环。具有广泛的抑菌效应，如革兰氏阳性菌、革兰氏阴性菌、分支杆菌、真菌等。早期研究发现地喹氯铵具有抗肿瘤作用，能延长膀胱癌或者结肠癌小鼠的生存时间，并且比常用的化疗药物如氨甲喋呤、顺铂等更有效。是一种潜在的抗肿瘤药物。目前尚未有报道地喹氯铵对神经胶质瘤的影响。本实验前期研究也发现地喹氯铵能够明显抑制体外培养的神经胶质瘤细胞的生长。因此，本研究观察了地喹氯铵在体内和体外对神经胶质瘤的作用，并探讨其可能的作用机制。首先我们观察了地喹氯铵对人神经胶质瘤细胞系SHG44、U251、U87细胞生长增殖的影响。MTT试验结果显示，，地喹氯铵能明显的抑制人神经胶质瘤细胞SHG44、U251、U87生长增殖，并呈剂量及时间依赖效应。同TMZ阳性对照组比较，DECA能在低药物浓度时对肿瘤细胞增殖有强烈的抑制作用。DECA对SHG44、U251、U87细胞的半数抑制浓度IC50为132.2±8.7μM、85.9±9.2μM和71.5±6.5μM，分别低于TMZ的508.3±40.6μM、468.5±39.7μM和381.9±41.3μM。提示DECA可能会较TMZ有更好的抑制神经胶质瘤细胞增殖的作用。采用SHG44细胞制备BALB/C裸鼠神经胶质瘤模型，观察地喹氯铵对肿瘤生长的影响，测量肿瘤体积，绘制肿瘤生长曲线。结果显示，与阴性对照组比较，地喹氯铵（10mg/kg）和TMZ（50mg/kg）能明显的抑制小鼠肿瘤生长（p0.01，p0.05），具有统计学意义；而地喹氯铵组与TMZ组小鼠肿瘤体积无明显差别（p=0.406）；SHG44细胞接种BALB/C裸鼠后30天处死小鼠剥离肿瘤，根据瘤重计算抑瘤率，DECA（10mg/kg）和TMZ（50mg/kg）的抑瘤率分别为52.05±3.68和34.56±5.29，提示地喹氯铵具有良好的抗肿瘤活性。这与体外的细胞实验结果是一致的。同时本研究通过基因组DNA电泳、DAPI染色和TUNEL染色发现，地喹氯铵能诱导体外培养的人胶质瘤细胞SHG44细胞的凋亡，提示地喹氯铵抑制肿瘤细胞的增殖可能是通过促进肿瘤细胞凋亡来实现的。为此我们通过生物信息学方法筛选出神经胶质瘤相关的发病基因，通过RT-PCR方法对检测地喹氯铵在体外对神经胶质瘤细胞表达这些基因mRNA水平上的差异，对这些基因进行初步筛选。结果显示25个基因中有6个基因mRNA水平表达出现差异，分别为NFKB2、HRAS、NF1、CBL、RAF1和Bcl-2基因，提示DECA有可能通过影响以上基因的表达发挥促进肿瘤细胞凋亡的作用。为了进一步探讨DECA促进肿瘤细胞凋亡的机制，我们采用了WB方法，分别检测了DECA刺激的SHG44细胞和荷瘤鼠肿瘤细胞中的凋亡相关蛋白表达。结果显示，地喹氯铵组SHG44细胞中Bcl-2蛋白表达降低，而Caspase-3、Bax蛋白表达增高；同时肿瘤中也观察到DECA能下调Bcl-2蛋白表达，上调Caspase-3、Bax蛋白表达。提示DECA可能通过调控凋亡转导通路中的Bcl-2蛋白和Caspase-3蛋白发挥促肿瘤细胞凋亡作用。综上所述，地喹氯铵在体外和体内实验中均能抑制神经胶质瘤细胞的生长，此作用可能是通过促进肿瘤细胞凋亡来实现的。对于细胞凋亡信号通路相关蛋白的检测结果表明地喹氯铵可能通过下调Bcl-2蛋白，上调Caspase-3、Bax蛋白途径发挥促进胶质瘤细胞凋亡的作用。地喹氯铵对神经胶质瘤的作用机制还需在以后的研究中进一步探讨。
[Abstract]:Glioma is the most common intracranial tumors, which is characterized by short course, difficult to cure, high fatality rate. The glioma treatment including surgical resection, radiotherapy, chemotherapy, immunotherapy, molecular targeted therapy. Surgical treatment is the first choice for the treatment of the disease, but because glial with its own characteristics, the fuzzy boundaries of the tumor tissue and normal brain tissue, not easy to identify, and the tumor growth can be extended to multiple important functional areas, the surgical treatment of complete excision difficult. Simple surgical treatment is often difficult to reach the number of tumor cells to reduce the immune system to kill the tumor cells, therefore the effect surgical treatment is often poor, MRI examination after surgery showed imaging can reach only about 60% of the total resection of glioma patients. So the glioma treatment is at hand Surgical resection is a comprehensive treatment strategy based on radiotherapy, chemotherapy, immunotherapy and molecular targeted therapy. However, there is still no breakthrough in the treatment of glioma. Exploring the innovative diagnosis and treatment technology of glioma has always been a research focus in Department of neurosurgery.
The alkylating agent temozolomide (temozolomide, TMZ) in the clinical application, the malignant glioma chemotherapy has made significant progress, people begin to attach importance to the chemotherapy drug anti glioma effect. Although the application of TMZ makes the glioma treatment effect is significantly improved, but because of the difficult to pass the blood brain barrier, tumor and surrounding tissue space static pressure higher effective drug concentration resulted in a decrease in drug resistance, long-term medication caused by tumor cells, the adverse reaction of the body, the molecular genetics changes in treating difficulties, will adversely affect the treatment of glioma. Therefore, the treatment of glioma is still facing many difficulties. Looking for a low toxicity, can target cancer drug, has been the development of anti-cancer drugs.
Dequalinium chloride (Dequalinium chloride DECA) is a synthetic quinoline derivatives, containing two isolated aromatic heterocyclic quinoline. Has the antibacterial effect of broad, such as gram positive bacteria, gram negative bacteria, mycobacteria, fungi and so on. Earlier research found that Dequalinium chloride has anti-tumor effect, can prolong the bladder or colon cancer the survival time of mice, and compared with the commonly used chemotherapy drugs such as methotrexate, cisplatin and other more effective. As a potential antitumor drug. Yet there are reports of Dequalinium chloride on glioma. The preliminary experiment research also found that glioma cells Dequalinium chloride can inhibit in vitro the growth. Therefore, this study is to observe the effect of Dequalinium chloride on glioma cells in vivo and in vitro, and to explore its possible mechanism.
First, we observed the Dequalinium chloride on human glioma cell lines SHG44, U251,.MTT test results of the proliferation of U87 cells showed that Dequalinium chloride can inhibit human glioma cell SHG44, obviously U251, U87 proliferation, and the effect was dose and time dependent manner. Compared with TMZ positive group. DECA can have a strong inhibitory effect of.DECA on SHG44, U251 on the proliferation of tumor cells in low concentration, half inhibitory concentration IC50 U87 cells was 132.2 + 8.7 M 85.9 + 9.2 M and 71.5 + 6.5 M, TMZ were less than 508.3 + 40.6 M, 39.7 M and 468.5. 381.9 + 41.3 M. suggest that DECA may be better than TMZ inhibits the proliferation of glioma cells. BALB/C glioma model in nude mice with SHG44 cells, observe the effect of Dequalinium chloride on tumor growth, tumor volume was measured and the tumor growth curve. The results show that with the female The control group, Dequalinium chloride (10mg/kg) and TMZ (50mg/kg) can inhibit tumor growth significantly (P0.01, P0.05), with statistical significance; and Dequalinium chloride group and TMZ group were no significant difference in tumor volume (p=0.406); tumor mice were sacrificed 30 days after inoculation of BALB/C SHG44 cells in nude mice. According to the tumor weight inhibition rate was calculated, DECA (10mg/kg) and TMZ (50mg/kg) and the inhibition rates were 52.05 + 3.68 and 34.56 + 5.29, suggesting that Dequalinium chloride has a good anti-tumor activity. This is consistent with in vitro results.
At the same time this research through genomic DNA electrophoresis, DAPI staining and TUNEL staining showed that Dequalinium chloride can induce apoptosis of cultured human glioma cells SHG44 cells, suggesting that Dequalinium chloride could inhibit the proliferation of tumor cells by promoting tumor cell apoptosis. Therefore we through bioinformatics and identified genes in neural pathogenesis glioma related, by the RT-PCR method for the detection of Dequalinium chloride in vitro on glioma cells. These genes are differentially expressed on the level of mRNA, these genes were screened. The results showed that the expression of 6 genes mRNA level difference of 25 genes, respectively NFKB2, HRAS, NF1, CBL, and RAF1 the Bcl-2 gene, suggesting that DECA may play a role in promoting tumor cell apoptosis by affecting the expression of these genes.
In order to further explore the mechanism of DECA induced apoptosis of tumor cells, we used the WB method, were detected in DECA stimulated SHG44 cells and tumor cells in tumor bearing mice apoptosis related protein expression. The results showed that the decreased expression of Bcl-2 protein in SHG44 cells of Dequalinium chloride group and Caspase-3, Bax protein expression and tumor; also observed that DECA can decrease the expression of Bcl-2, upregulation of Caspase-3, expression of Bax protein. It indicates that DECA may through Bcl-2 and Caspase-3 proteins in the regulation of apoptosis pathway to promote apoptosis of tumor cells.
In summary, Dequalinium chloride could inhibit glioma cells in vitro and in vivo growth, this effect may be through the promotion of tumor cell apoptosis. The results of detection for signal pathway of apoptosis related proteins that Dequalinium through down-regulation of Bcl-2 protein, up-regulated Caspase-3, Bax protein pathway play roles in promoting apoptosis glioma cells. The mechanism of Dequalinium chloride on glioma still need to be further studied in future research.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.4

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