本文關(guān)鍵詞： 羥基芫花素 芹菜素 協(xié)同配伍 膠質(zhì)瘤 凋亡　出處：《大連醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：初步探索羥基芫花素與芹菜配伍協(xié)同抗膠質(zhì)瘤作用及其分子機(jī)制 方法：MTT法檢測經(jīng)不同濃度的芹菜素、羥基芫花素、羥基芫花素+芹菜素（配伍組）處理不同時間后的C6膠質(zhì)瘤細(xì)胞增殖情況，初步判定各組藥物抑制C6膠質(zhì)瘤增殖的時間依賴、濃度依賴特點(diǎn)。用AO/EB（acridine orange/ethidiumbromide，，吖啶橙/溴化乙錠）和DAPI（4,6-diamidino-2-phenylindole，4,6-二脒基-2-苯基吲哚）熒光染色的方法檢測核固縮等細(xì)胞凋亡形態(tài)變化；應(yīng)用掃描電鏡（transmission electron microscope，TEM）和JC-1熒光探針的方法從形態(tài)和功能的角度檢測各組藥物對線粒體結(jié)構(gòu)和膜電位的損傷情況；單細(xì)胞凝膠電泳（single cell gel eletrophoresis，SCGE)實驗檢測細(xì)胞DNA損傷；流式細(xì)胞儀檢測各組藥物對C6細(xì)胞的周期阻滯作用。在分子機(jī)制研究方面，采用Western blot考察了各組藥物對線粒體凋亡通路相關(guān)抗凋亡蛋白（Bcl-2、Bcl-xl）和凋亡誘導(dǎo)蛋白（Bax、Bak、Bid、Bad）表達(dá)的影響；利用放免試劑盒測定了經(jīng)各組藥物處理后C6膠質(zhì)瘤細(xì)胞上清液中TNF-的濃度；Caspase活性測定試劑盒檢測各組藥物對腫瘤細(xì)胞Caspase-3和8酶活性的影響。 結(jié)果：羥基芫花素和芹菜素在總藥物濃度為25μM時對C6膠質(zhì)瘤細(xì)胞均有一定的抗增殖效應(yīng)，其中羥基芫花素的抗膠質(zhì)瘤效應(yīng)稍強(qiáng)于芹菜素；當(dāng)二者配伍聯(lián)合應(yīng)用后，聯(lián)合用藥組的抗膠質(zhì)瘤效應(yīng)明顯強(qiáng)于各單用藥組；AO/EB染色、DAPI染色結(jié)果表明羥基芫花素和芹菜素是通過誘導(dǎo)腫瘤細(xì)胞凋亡的方式達(dá)到抗增殖效應(yīng)；進(jìn)一步機(jī)制研究表明，羥基芫花素對線粒體結(jié)構(gòu)和功能（線粒體膜電位）造成明顯的損傷，聯(lián)合用藥組表現(xiàn)出明顯的協(xié)同效應(yīng)；SCGE檢測到各處理組中腫瘤細(xì)胞DNA損傷。細(xì)胞周期分析表明，羥基芫花素具有明顯的S期阻滯效應(yīng)，而芹菜素對腫瘤細(xì)胞周期無明顯的阻滯效應(yīng)；進(jìn)一步分子機(jī)制研究表明，相對于對照組，羥基芫花素和芹菜素能明顯增強(qiáng)腫瘤細(xì)胞TNF-的表達(dá)、增強(qiáng)Caspase活化，當(dāng)聯(lián)合應(yīng)用后二者表現(xiàn)出明顯的協(xié)同效應(yīng)。但是Western blot結(jié)果分析表明，羥基芫花素和芹菜素對線粒體凋亡通路某些關(guān)鍵蛋白表達(dá)的影響具有無明顯的協(xié)同效應(yīng)；藥物毒性初步評估結(jié)果表明，在一定濃度下，羥基芫花素對高分化的PC12細(xì)胞系無明顯毒性效應(yīng)（IC50㧐120μM）。 結(jié)論：羥基芫花素具有一定的抗膠質(zhì)瘤作用，當(dāng)羥基芫花素與芹菜素聯(lián)合應(yīng)用后，二者表現(xiàn)出明顯的協(xié)同增效抗膠質(zhì)瘤效應(yīng)；羥基芫花素和芹菜素通過線粒體損傷、DNA損傷、細(xì)胞周期S期阻滯，啟動線粒體凋亡通路等機(jī)制達(dá)到抗膠質(zhì)瘤作用。本研究首次證明了羥基芫花素聯(lián)合芹菜素的巨大抗膠質(zhì)瘤潛在價值并為進(jìn)一步動物體內(nèi)研究提供了一定的理論依據(jù)。
[Abstract]:Objective: to explore the synergistic effect of hydroxygenkwa with celery on glioma and its molecular mechanism. Methods the proliferation of C6 glioma cells treated with different concentrations of apigenin, hydroxygenkapenin and hydroxygenkapenin (compatibility group) for different time was detected by MTT assay. The time and concentration dependence of drug inhibition on the proliferation of C6 gliomas was preliminarily determined. The morphological changes of nuclear pyknosis were detected by fluorescence staining of AO/EB(acridine orange / ethidium rodede (acridine orange / ethidium bromide) and DAPIX 46-diamidino-2-phenylindoledol (46-diamidino-2-phenylindoledol). The morphology and function of mitochondrial structure and membrane potential of each group were examined by scanning electron microscope transmission electron microscopetem and JC-1 fluorescence probe, and single cell gel eletrophoresis-SCGE assay was used to detect cell DNA damage. The cell cycle arrest of C6 cells was detected by flow cytometry. In the study of molecular mechanism, Western blot was used to investigate the effects of each group on the expression of Bcl-2Bcl-xl) and Bax-induced protein Baxy BakBidadad. The concentration of TNF- in the supernatant of C6 glioma cells was determined by radioimmunoassay kit. The effects of various drugs on the activities of Caspase-3 and 8 enzymes in the supernatant of C6 glioma cells were measured. Results: both hydroxygenkaphnin and apigenin could inhibit the proliferation of C6 glioma cells at a concentration of 25 渭 M, and the anti-glioma effect of hydroxygenkwa was slightly stronger than that of apigenin. The anti-glioma effect of the combined treatment group was significantly stronger than that of each single drug group by AO-EB staining and DAPI staining. The results showed that hydroxygenkaphnin and apigenin could induce the apoptosis of tumor cells to achieve the anti-proliferation effect. Hydroxygenkwa caused obvious damage to mitochondrial structure and function (mitochondrial membrane potential), and the combined treatment group showed obvious synergistic effect. Cell cycle analysis showed that the tumor cell DNA damage was detected in each treatment group. Hydroxy Daphne genkwa has obvious S phase arrest effect, while apigenin has no obvious blocking effect on tumor cell cycle. Hydroxygenkwa and apigenin could significantly enhance the expression of TNF- and the activation of Caspase in tumor cells, but the results of Western blot analysis showed that the expression of TNF- and the activation of TNF- in tumor cells were significantly increased after combined treatment. There was no significant synergistic effect of hydroxygenkwa and apigenin on the expression of some key proteins in mitochondrial apoptotic pathway. Hydroxygenkwanin has no toxic effect on highly differentiated PC12 cell line. 120 渭 m. Conclusion: hydroxygenkwa has a certain anti-glioma effect. When the combination of hydroxygenkwa and apigenin is used, both of them have synergistic effect on glioma, and hydroxygenkaphnin and apigenin damage DNA through mitochondria. Cell cycle S phase arrest, This study demonstrated the potential value of hydroxygenkaphnin combined with apigenin in anti-glioma and provided a theoretical basis for further study in vivo.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R739.41

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