本文選題：骨肉瘤　切入點(diǎn)：匹莫齊特　出處：《深圳大學(xué)》2017年碩士論文

【摘要】：背景:骨肉瘤(Osteosarcoma,OS)是原發(fā)性惡性骨腫瘤的一類,在青少年及兒童群體中發(fā)生率為2~5/100萬(wàn)人,易產(chǎn)生遠(yuǎn)端轉(zhuǎn)移,致其患者預(yù)后效果較差。研究報(bào)道STAT3信號(hào)通路的持續(xù)激活對(duì)骨肉瘤細(xì)胞的增殖、存活、藥物耐藥性等產(chǎn)生直接影響,故而靶向抑制STAT3信號(hào)通路的藥物將可成為抑制骨肉瘤細(xì)胞增殖的有效方法。前期研究工作發(fā)現(xiàn),用于治療精神病的藥物匹莫齊特(Pimozide)能抑制細(xì)胞內(nèi)STAT3信號(hào)通路的活化,進(jìn)而抑制肝癌細(xì)胞株MHCC-97L、Hep3B的生長(zhǎng)增殖。為此,本研究探討匹莫齊特抗骨肉瘤的分子機(jī)制。目的:研究匹莫齊特對(duì)骨肉瘤細(xì)胞中STAT3信號(hào)通路活化的影響,匹莫齊特對(duì)人骨肉瘤細(xì)胞是否具有抗癌作用及其相關(guān)作用機(jī)制。方法:(1)培養(yǎng)人骨肉瘤細(xì)胞系;匹莫齊特加藥處理骨肉瘤細(xì)胞;(2)采用蛋白免疫印跡實(shí)驗(yàn)分析STAT3通路有關(guān)蛋白p-STAT3的表達(dá)水平;通過(guò)RT-qPCR檢測(cè)STAT3通路下游基因的轉(zhuǎn)錄表達(dá)水平;利用Dual-Luciferase報(bào)告基因系統(tǒng)檢測(cè)STAT3通路的激活程度;(3)通過(guò)克隆形成實(shí)驗(yàn),球體形成實(shí)驗(yàn)和流式檢測(cè)骨肉瘤細(xì)胞的增殖、凋亡和細(xì)胞周期變化;(4)采用活性氧檢檢測(cè)試劑盒測(cè)定骨肉瘤細(xì)胞內(nèi)ROS含量;(5)通過(guò)細(xì)胞增殖實(shí)驗(yàn)觀察過(guò)表達(dá)CAT基因后對(duì)匹莫齊特效用的影響;(6)開(kāi)展染色質(zhì)免疫共沉淀實(shí)驗(yàn)分析CAT基因與STAT3的關(guān)系。結(jié)果:(1)匹莫齊特可以降低骨肉瘤細(xì)胞內(nèi)STAT3信號(hào)活性,主要表現(xiàn)為降低信號(hào)激活標(biāo)志物p-STAT3表達(dá)水平、下調(diào)STAT3信號(hào)下游基因MYC、BCL-xl和MCL-1的轉(zhuǎn)錄表達(dá)水平,并且抑制STAT3-Luc報(bào)告基因的活性;(2)匹莫齊特抑制骨肉瘤細(xì)胞系U2OS、MG-63與SW1353細(xì)胞的增殖和骨肉瘤細(xì)胞的克隆形成和球體形成能力,表明匹莫齊特能抑制骨肉瘤細(xì)胞的干性能力,主要表現(xiàn)為下調(diào)干性相關(guān)因子Sox2、Nanog和c-Myc蛋白表達(dá)、以及增強(qiáng)細(xì)胞對(duì)5-FU誘導(dǎo)增殖抑制效果和降低細(xì)胞內(nèi)側(cè)群細(xì)胞(Side population,SP)的比例;(3)匹莫齊特可誘導(dǎo)骨肉瘤細(xì)胞發(fā)生凋亡,并使細(xì)胞的G0/G1期產(chǎn)生阻滯;(4)匹莫齊特能抑制細(xì)胞Erk信號(hào)激活影響骨肉瘤細(xì)胞的增殖活性,表現(xiàn)為下調(diào)p-Erk蛋白的表達(dá);Erk通路抑制劑無(wú)法增強(qiáng)匹莫齊特對(duì)細(xì)胞的增殖抑制效果;(5)匹莫齊特抑制細(xì)胞內(nèi)CAT基因表達(dá)而促進(jìn)ROS產(chǎn)生,但對(duì)SOD1、SOD2和GPX1等基因表達(dá)沒(méi)有影響;CAT過(guò)表達(dá)后能減弱匹莫齊特對(duì)骨肉瘤細(xì)胞的增殖抑制作用;(6)生物信息學(xué)推測(cè)CAT基因的調(diào)控序列中可能含有兩個(gè)STAT3結(jié)合的潛在位點(diǎn);ChIP實(shí)驗(yàn)結(jié)果顯示STAT3蛋白可結(jié)合在CAT基因的啟動(dòng)子區(qū)。結(jié)論:本課題研究發(fā)現(xiàn)匹莫齊特能有效地抑制骨肉瘤細(xì)胞內(nèi)STAT3信號(hào)活化,對(duì)骨肉瘤細(xì)胞具有抗癌作用;并提出匹莫齊特的抗癌分子機(jī)制是通過(guò)抑制骨肉瘤細(xì)胞中STAT3信號(hào)活性而降低CAT基因的表達(dá),進(jìn)而產(chǎn)生大量ROS導(dǎo)致細(xì)胞產(chǎn)生增殖抑制和細(xì)胞凋亡。匹莫齊特可作為STAT3的潛在靶向抑制劑,有望成為治療骨肉瘤的潛在藥物或者先導(dǎo)化合物,為臨床應(yīng)用奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R738

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本文編號(hào)：1730495