本文關鍵詞： miR-16 miR-21 槐果堿 血根堿 增殖、凋亡　出處：《西安電子科技大學》2015年碩士論文　論文類型：學位論文

【摘要】：癌癥是威脅人類生命的一種嚴重病癥。癌癥治愈的幾率遠小于其他疾病,幾乎是不治之癥。癌癥治療一直是醫(yī)學領域最為活躍的研究課題之一。如果癌癥能夠提早發(fā)現(xiàn)和治療,治愈的幾率也將大幅提高。研究發(fā)現(xiàn),在腫瘤細胞中miR-21特異性高表達,miR-16低表達顯示這兩種microRNA可作為癌癥治療的潛在靶標。此外,現(xiàn)有研究發(fā)現(xiàn),一些天然藥物能夠改變microRNA的表達,從而影響腫瘤細胞的生長、增殖或分化過程。本文主要篩選能夠調控miR-21和miR-16表達的天然藥物以及研究它們對腫瘤細胞增殖和凋亡的影響。因此,本文首選通過螢火蟲熒光素酶基因的3’UTR插入與miR-21或者miR-16完全互補的序列,構建成響應miR-21或miR-16表達的miR-21熒光報告基因和miR-16熒光報告基因,通過檢測熒光變化來間接反映miR-21和mi R-16的表達量。熒光素酶實驗發(fā)現(xiàn)槐果堿上調miR-21的表達,血根堿下調miR-16的表達。首先,對miR-21和槐果堿進行細胞和動物機制的研究。具體的實驗有實時定量PCR、免疫印跡實驗(Western blot)、細胞增殖檢測實驗(CCK-8)等。通過實時定量PCR證明槐果堿上調miR-21的表達;通過免疫印跡法證明槐果堿下調mi R-21的靶基因PTEN的表達;通過信號通路實驗,發(fā)現(xiàn)槐果堿通過NF-KB和P38MAPK信號通路上調miR-21的表達;細胞增殖檢測實驗發(fā)現(xiàn)anti-miR-21 RNA和槐果堿能夠共同抑制A549細胞的增殖�；惫麎A裸鼠腫瘤治療研究,發(fā)現(xiàn)槐果堿對腫瘤生長有抑制作用。其次,miR-16和血根堿進行了細胞和動物機制的研究。具體的實驗有實時定量PCR、免疫印跡實驗、caspase-3活性檢測實驗和裸鼠腫瘤治療實驗。實時定量PCR實驗發(fā)現(xiàn)了血根堿下調miR-16的表達;免疫印跡實驗發(fā)現(xiàn)了血根堿下調Bcl-2蛋白;通過caspase-3活性檢測實驗,表明了miR-16和血根堿協(xié)同激活caspase-3的表達;裸鼠成瘤實驗發(fā)現(xiàn)血根堿能夠抑制裸鼠腫瘤生長,即血根堿具有抗癌作用�？傊�,通過研究發(fā)現(xiàn)槐果堿通過靶向miR-21對腫瘤細胞增殖有抑制作用,血根堿通過靶向miR-16促進細胞凋亡,因而槐果堿和血根堿可以作為潛在的藥物用于腫瘤的治療。
[Abstract]:Cancer is a serious disease that threatens human life. Cancer is much less likely to be cured than other diseases and almost incurable. Cancer treatment has been one of the most active research topics in medicine. The likelihood of cure will also be significantly increased. Studies have found that the high expression of miR-21 specifically in cancer cells suggests that the two types of microRNA may be potential targets for cancer treatment. Some natural drugs can alter the expression of microRNA, thus affecting the growth of tumor cells. Proliferation or differentiation. In this paper, we select natural drugs that can regulate the expression of miR-21 and miR-16 and study their effects on the proliferation and apoptosis of tumor cells. In this paper, we first constructed the miR-21 fluorescent reporter gene and the miR-16 fluorescent reporter gene in response to the expression of miR-21 or miR-16 by inserting 3UTR of the luciferase gene of the firefly worm into a sequence that is completely complementary to miR-21 or miR-16. Luciferase assay showed that Sophorine upregulated the expression of miR-21 and blood root alkaloids down-regulated the expression of miR-16. The cellular and animal mechanisms of miR-21 and sophorline were studied. The specific experiments were real-time quantitative PCRs, Western blotl assay and cell proliferation assay (CCK-8). It was proved by real-time quantitative PCR that Sophorine upregulated the expression of miR-21. The results showed that Sophorine down-regulated the expression of PTEN in target gene of miR-21 by Western blot, and up-regulated the expression of miR-21 through NF-KB and p38 MAPK signaling pathway through signal pathway experiment. Cell proliferation assay showed that anti-miR-21 RNA and sophorline could inhibit the proliferation of A549 cells. It was found that Sophorine had inhibitory effect on tumor growth. Secondly, the cellular and animal mechanisms of miR-16 and root alkaloid were studied. The specific experiments included real-time quantitative PCR, Western blot assay, caspase-3 activity detection and tumor therapy in nude mice. Real-time quantitative PCR assay showed that root alkaloids down-regulated the expression of miR-16; Western blotting showed that root alkaloids down-regulated Bcl-2 protein; caspase-3 activity test showed that miR-16 and root alkaloids co-activated caspase-3 expression; in nude mice tumorigenesis, root alkaloids inhibited tumor growth in nude mice. In conclusion, it was found that Sophorine inhibits the proliferation of tumor cells through targeted miR-21, and root alkaloids promote cell apoptosis through targeted miR-16. Therefore, Sophorine and root alkaloids can be used as potential drugs for the treatment of tumors.
【學位授予單位】：西安電子科技大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R730.5

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