【摘要】：實(shí)驗(yàn)?zāi)康?觀察帕比司他單用及其聯(lián)合STF083010對食管癌細(xì)胞生長增殖的影響,并初步探討其作用機(jī)制。實(shí)驗(yàn)方法:采用CCK8法檢測藥物對食管癌細(xì)胞的生長抑制作用;PI單染法檢測細(xì)胞周期;Annexin V/PI雙染色法檢測細(xì)胞凋亡;Western blot和Quantitative RT-PCR檢測藥物作用細(xì)胞后蛋白及基因表達(dá)水平的變化;采用激光共聚焦法檢測藥物作用細(xì)胞后對內(nèi)質(zhì)網(wǎng)形態(tài)的影響;構(gòu)建裸鼠移植瘤模型檢測藥物在體內(nèi)抗食管癌的效果。實(shí)驗(yàn)結(jié)果:帕比司他有效抑制對傳統(tǒng)化療藥物不敏感的Kyse450和Kyse510食管癌細(xì)胞的生長。帕比司他通過增加組蛋白H3和α-微管蛋白乙�；�,上調(diào)p21和降低c-myc的表達(dá)而誘導(dǎo)細(xì)胞凋亡和G2/M期周期阻滯。同時(shí),帕比司他誘導(dǎo)細(xì)胞產(chǎn)生內(nèi)質(zhì)網(wǎng)應(yīng)激,激活I(lǐng)RE1-Xbp1信號通路。帕比司他與IRE1核酸內(nèi)切酶抑制劑STF083010聯(lián)用顯著抑制食管癌細(xì)胞增殖,大部分兩藥聯(lián)合作用指數(shù)(CI)1。與單用帕比司他治療組相比,帕比司他與STF083010聯(lián)用組顯著抑制IRE1-Xbp1促存活信號通路,抑制依賴HDAC6的聚集小體降解途徑,上調(diào)內(nèi)質(zhì)網(wǎng)應(yīng)激凋亡標(biāo)志物DDIT3基因的表達(dá),進(jìn)而下調(diào)Bcl-xL/Bax的比例,激活caspase級聯(lián)反應(yīng),增強(qiáng)PARP切割,從而誘導(dǎo)細(xì)胞凋亡。在裸鼠移植瘤模型中,STF083010顯著增強(qiáng)帕比司他介導(dǎo)的腫瘤生長抑制及腫瘤細(xì)胞壞死的數(shù)量。結(jié)論:IRE1核酸內(nèi)切酶抑制劑STF083010在體內(nèi)外都能夠顯著促進(jìn)帕比司他對食管癌增殖的抑制作用,并且促進(jìn)帕比司他誘導(dǎo)的食管癌細(xì)胞發(fā)生凋亡。
[Abstract]:Aim: to observe the effect of pabestat alone and its combination with STF083010 on the growth and proliferation of esophageal cancer cells and to explore its mechanism. Methods: CCK8 assay was used to detect the growth inhibition of esophageal cancer cells, PI single staining method was used to detect cell cycle, and Annexin V/PI double staining method was used to detect apoptosis. Western blot and Quantitative RT-PCR were used to detect the changes of protein and gene expression, and laser confocal method was used to detect the morphology of endoplasmic reticulum (ER). A nude mouse model of transplanted tumor was established to detect the effect of drugs on esophageal cancer in vivo. Results: Papillast effectively inhibited the growth of Kyse450 and Kyse510 esophageal cancer cells insensitive to traditional chemotherapeutic agents. Pabestar induces apoptosis and G 2 / M cycle arrest by increasing the acetylation level of histone H3 and 偽 -tubulin, up-regulating p21 and decreasing the expression of c-myc. At the same time, paracetamil induces endoplasmic reticulum stress and activates IRE1-Xbp1 signaling pathway. The combination of pabestat and IRE1 endonuclease inhibitor STF083010 significantly inhibited the proliferation of esophageal cancer cells. The combined action index of most of the two drugs was (CI) 1. Compared with the control group, the combination of paracetamol and STF083010 significantly inhibited the IRE1-Xbp1 survival signaling pathway, inhibited the aggregation body degradation pathway dependent on HDAC6, and up-regulated the expression of DDIT3 gene, the endoplasmic reticulum stress marker of apoptosis. Furthermore, the ratio of Bcl-xL/Bax was down-regulated, caspase cascade was activated, PARP cleavage was enhanced, and apoptosis was induced. In nude mice model of transplanted tumor, STF083010 significantly enhanced the amount of tumor growth inhibition and tumor cell necrosis mediated by paracetamil. Conclusion: IRE1 endonuclease inhibitor STF083010 can significantly inhibit the proliferation of esophageal carcinoma and induce apoptosis of esophageal cancer cells induced by pabestatin in vitro and in vivo.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.1

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