本文選題：神經(jīng)膠質(zhì)瘤　切入點(diǎn)：地喹氯銨　出處：《吉林大學(xué)》2014年博士論文

【摘要】：神經(jīng)膠質(zhì)瘤是最常見(jiàn)的顱內(nèi)腫瘤，其特點(diǎn)是病程短、治愈難、病死率高。目前神經(jīng)膠質(zhì)瘤的治療方式主要包括了手術(shù)切除、放射治療、化學(xué)治療、免疫治療、分子靶向治療等。手術(shù)治療為該病治療首選方法，但由于神經(jīng)膠質(zhì)瘤自身特點(diǎn)，腫瘤組織與正常腦組織界限模糊、不易辨別，且腫瘤可生長(zhǎng)延伸至多個(gè)重要功能區(qū)域，使得手術(shù)治療徹底切除病灶難度很大。單純性手術(shù)治療往往很難達(dá)到使腫瘤細(xì)胞數(shù)量減少到機(jī)體免疫系統(tǒng)能消滅腫瘤細(xì)胞的程度，因此手術(shù)治療效果往往不佳，手術(shù)后的MRI復(fù)查顯示腦膠質(zhì)瘤患者中僅60%左右的可以達(dá)到影像學(xué)全切除。所以目前神經(jīng)膠質(zhì)瘤的治療方式是以手術(shù)切除為基礎(chǔ)，結(jié)合放射治療、化學(xué)治療、免疫治療、分子靶向治療等方法形成的綜合治療策略。但膠質(zhì)瘤治療仍缺乏突破性進(jìn)展。探索膠質(zhì)瘤創(chuàng)新診療技術(shù)，一直是神經(jīng)外科學(xué)的研究熱點(diǎn)。 烷化劑替莫唑胺（temozolomide，TMZ）在臨床上的應(yīng)用，使惡性腦膠質(zhì)瘤患者化療取得了重大進(jìn)展，人們重新開(kāi)始重視化療藥物的抗膠質(zhì)瘤作用。但盡管TMZ的應(yīng)用使得神經(jīng)膠質(zhì)瘤的治療效果有了顯著的改善，但因其難以通過(guò)血腦屏障、腫瘤及周邊組織間隙靜水壓高等導(dǎo)致的藥物有效濃度降低，長(zhǎng)期用藥引起腫瘤細(xì)胞產(chǎn)生的耐藥性，對(duì)機(jī)體的不良反應(yīng)，分子遺傳學(xué)改變導(dǎo)致的治療困難等，均會(huì)對(duì)神經(jīng)膠質(zhì)瘤治療產(chǎn)生不利影響。因此，膠質(zhì)瘤的治療仍面臨許多困難。尋找具有低毒副作用、能夠靶向作用于腫瘤的藥物，一直是抗腫瘤藥物研發(fā)的目標(biāo)。 地喹氯銨（dequalinium chloride，DECA）是人工合成的喹啉衍生物，含有兩個(gè)孤立的喹啉芳雜環(huán)。具有廣泛的抑菌效應(yīng)，如革蘭氏陽(yáng)性菌、革蘭氏陰性菌、分支桿菌、真菌等。早期研究發(fā)現(xiàn)地喹氯銨具有抗腫瘤作用，能延長(zhǎng)膀胱癌或者結(jié)腸癌小鼠的生存時(shí)間，并且比常用的化療藥物如氨甲喋呤、順鉑等更有效。是一種潛在的抗腫瘤藥物。目前尚未有報(bào)道地喹氯銨對(duì)神經(jīng)膠質(zhì)瘤的影響。本實(shí)驗(yàn)前期研究也發(fā)現(xiàn)地喹氯銨能夠明顯抑制體外培養(yǎng)的神經(jīng)膠質(zhì)瘤細(xì)胞的生長(zhǎng)。因此，本研究觀察了地喹氯銨在體內(nèi)和體外對(duì)神經(jīng)膠質(zhì)瘤的作用，并探討其可能的作用機(jī)制。 首先我們觀察了地喹氯銨對(duì)人神經(jīng)膠質(zhì)瘤細(xì)胞系SHG44、U251、U87細(xì)胞生長(zhǎng)增殖的影響。MTT試驗(yàn)結(jié)果顯示，，地喹氯銨能明顯的抑制人神經(jīng)膠質(zhì)瘤細(xì)胞SHG44、U251、U87生長(zhǎng)增殖，并呈劑量及時(shí)間依賴效應(yīng)。同TMZ陽(yáng)性對(duì)照組比較，DECA能在低藥物濃度時(shí)對(duì)腫瘤細(xì)胞增殖有強(qiáng)烈的抑制作用。DECA對(duì)SHG44、U251、U87細(xì)胞的半數(shù)抑制濃度IC50為132.2±8.7μM、85.9±9.2μM和71.5±6.5μM，分別低于TMZ的508.3±40.6μM、468.5±39.7μM和381.9±41.3μM。提示DECA可能會(huì)較TMZ有更好的抑制神經(jīng)膠質(zhì)瘤細(xì)胞增殖的作用。采用SHG44細(xì)胞制備BALB/C裸鼠神經(jīng)膠質(zhì)瘤模型，觀察地喹氯銨對(duì)腫瘤生長(zhǎng)的影響，測(cè)量腫瘤體積，繪制腫瘤生長(zhǎng)曲線。結(jié)果顯示，與陰性對(duì)照組比較，地喹氯銨（10mg/kg）和TMZ（50mg/kg）能明顯的抑制小鼠腫瘤生長(zhǎng)（p0.01，p0.05），具有統(tǒng)計(jì)學(xué)意義；而地喹氯銨組與TMZ組小鼠腫瘤體積無(wú)明顯差別（p=0.406）；SHG44細(xì)胞接種BALB/C裸鼠后30天處死小鼠剝離腫瘤，根據(jù)瘤重計(jì)算抑瘤率，DECA（10mg/kg）和TMZ（50mg/kg）的抑瘤率分別為52.05±3.68和34.56±5.29，提示地喹氯銨具有良好的抗腫瘤活性。這與體外的細(xì)胞實(shí)驗(yàn)結(jié)果是一致的。 同時(shí)本研究通過(guò)基因組DNA電泳、DAPI染色和TUNEL染色發(fā)現(xiàn)，地喹氯銨能誘導(dǎo)體外培養(yǎng)的人膠質(zhì)瘤細(xì)胞SHG44細(xì)胞的凋亡，提示地喹氯銨抑制腫瘤細(xì)胞的增殖可能是通過(guò)促進(jìn)腫瘤細(xì)胞凋亡來(lái)實(shí)現(xiàn)的。為此我們通過(guò)生物信息學(xué)方法篩選出神經(jīng)膠質(zhì)瘤相關(guān)的發(fā)病基因，通過(guò)RT-PCR方法對(duì)檢測(cè)地喹氯銨在體外對(duì)神經(jīng)膠質(zhì)瘤細(xì)胞表達(dá)這些基因mRNA水平上的差異，對(duì)這些基因進(jìn)行初步篩選。結(jié)果顯示25個(gè)基因中有6個(gè)基因mRNA水平表達(dá)出現(xiàn)差異，分別為NFKB2、HRAS、NF1、CBL、RAF1和Bcl-2基因，提示DECA有可能通過(guò)影響以上基因的表達(dá)發(fā)揮促進(jìn)腫瘤細(xì)胞凋亡的作用。 為了進(jìn)一步探討DECA促進(jìn)腫瘤細(xì)胞凋亡的機(jī)制，我們采用了WB方法，分別檢測(cè)了DECA刺激的SHG44細(xì)胞和荷瘤鼠腫瘤細(xì)胞中的凋亡相關(guān)蛋白表達(dá)。結(jié)果顯示，地喹氯銨組SHG44細(xì)胞中Bcl-2蛋白表達(dá)降低，而Caspase-3、Bax蛋白表達(dá)增高；同時(shí)腫瘤中也觀察到DECA能下調(diào)Bcl-2蛋白表達(dá)，上調(diào)Caspase-3、Bax蛋白表達(dá)。提示DECA可能通過(guò)調(diào)控凋亡轉(zhuǎn)導(dǎo)通路中的Bcl-2蛋白和Caspase-3蛋白發(fā)揮促腫瘤細(xì)胞凋亡作用。 綜上所述，地喹氯銨在體外和體內(nèi)實(shí)驗(yàn)中均能抑制神經(jīng)膠質(zhì)瘤細(xì)胞的生長(zhǎng)，此作用可能是通過(guò)促進(jìn)腫瘤細(xì)胞凋亡來(lái)實(shí)現(xiàn)的。對(duì)于細(xì)胞凋亡信號(hào)通路相關(guān)蛋白的檢測(cè)結(jié)果表明地喹氯銨可能通過(guò)下調(diào)Bcl-2蛋白，上調(diào)Caspase-3、Bax蛋白途徑發(fā)揮促進(jìn)膠質(zhì)瘤細(xì)胞凋亡的作用。地喹氯銨對(duì)神經(jīng)膠質(zhì)瘤的作用機(jī)制還需在以后的研究中進(jìn)一步探討。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.4

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉佳琪;;地喹氯銨含片含量測(cè)定分析[J];亞太傳統(tǒng)醫(yī)藥;2014年16期

2 吳嬋娟;黃碎錦;楊科;黃光亮;李寧;;微乳液相色譜法測(cè)定地喹氯銨噴霧劑中地喹氯銨的含量[J];廣東藥學(xué)院學(xué)報(bào);2010年01期

3 張崗;;地喹氯銨聯(lián)用潑尼松治療急性咽炎的臨床療效分析[J];中國(guó)醫(yī)藥指南;2014年15期

4 張琳;薛榮奇;董金鳳;;地喹氯銨噴霧劑治療急性咽炎30例[J];陜西醫(yī)學(xué)雜志;2006年10期

5 黃東青;何蓀蘭;史楚君;;地喹氯銨含片的含量測(cè)定及含量均勻度研究[J];廣東藥學(xué)院學(xué)報(bào);2007年03期

6 門螢;王小星;居瑞軍;田瑋;應(yīng)雪;姚紅娟;張燕;李若婧;呂萬(wàn)良;;高效液相紫外色譜法同時(shí)測(cè)定抗耐藥脂質(zhì)體中的表阿霉素、氨氯地平和地喹氯銨(英文)[J];Journal of Chinese Pharmaceutical Sciences;2010年04期

7 方文華;劉健;沈娟;張禮菊;;HPLC法測(cè)定地喹氯銨含片中地喹氯銨的含量[J];安徽醫(yī)藥;2011年07期

8 閆冬梅;賈琴;劉靜;石樹(shù)榮;;地喹氯銨噴霧劑聯(lián)合潰瘍散治療小兒阿弗他潰瘍療效觀察[J];人民軍醫(yī);2012年10期

9 黃蓉,朱景申,李素悅;地喹氯銨類似物構(gòu)效關(guān)系的研究進(jìn)展[J];醫(yī)藥導(dǎo)報(bào);2000年03期

10 肖紅俊,黃選兆,向友華;急性咽炎、扁桃體炎的細(xì)菌學(xué)特征及地喹氯銨口含片(泰樂(lè)奇)的療效觀察[J];臨床耳鼻咽喉科雜志;1998年07期

相關(guān)重要報(bào)紙文章 前1條

1 張石革 梁建華;嗓子冒“火”耐之何[N];醫(yī)藥經(jīng)濟(jì)報(bào);2002年

相關(guān)博士學(xué)位論文 前1條

1 于瑩;地喹氯銨對(duì)神經(jīng)膠質(zhì)瘤的作用及其機(jī)制研究[D];吉林大學(xué);2014年

本文編號(hào)：1724110