【摘要】：在哺乳動物中,核轉(zhuǎn)錄因子NF-κB是一類最常見、含量最豐富的核轉(zhuǎn)錄因子,NF-κB可被多種刺激因素激活,包括:基因毒性、炎癥因子以及氧化應(yīng)激。在腫瘤的發(fā)生發(fā)展中,活化的NF-κB與多種細(xì)胞過程相關(guān),包括:炎癥、轉(zhuǎn)化、細(xì)胞增殖、血管增生、細(xì)胞侵襲、轉(zhuǎn)移以及化學(xué)抵抗和抗輻射性。因此,化療藥物對NF-κB的抑制可作為治療腫瘤的潛在治療手段。黃芩素是從中國藥用植物黃芩的全草、根莖中提取出來的黃酮類化合物。已報道黃芩素具有抗氧化、抗炎、抗腫瘤、抗焦慮以及神經(jīng)保護(hù)等生物活性。本實(shí)驗(yàn)主要針對黃芩素以NF-1κB為靶點(diǎn)的抗炎抗腫瘤活性及其作用機(jī)制進(jìn)行研究。首先,通過熒光素酶報告基因?qū)嶒?yàn)檢測黃芩素對NF-κB轉(zhuǎn)錄活性的作用。發(fā)現(xiàn)在TNF-α刺激下,黃芩素可濃度依賴型抑制NF-κB轉(zhuǎn)錄活性。MTT實(shí)驗(yàn)證明了黃芩素對HeLa細(xì)胞沒有明顯的細(xì)胞毒性。通過免疫印跡法檢測了黃芩素對IκBα、p-IκBα、p65蛋白表達(dá)的作用。結(jié)果發(fā)現(xiàn),黃芩素可以通過抑制細(xì)胞質(zhì)內(nèi)IκBα的磷酸化與降解來抑制HeLa細(xì)胞中TNF-α刺激下p65的核轉(zhuǎn)移。通過免疫熒光法再次驗(yàn)證了黃芩素能顯著抑制TNF-α刺激下P65進(jìn)入細(xì)胞核,從而抑制了 NF-κB與其啟動子的結(jié)合。通過免疫印跡法檢測黃岑素對NF-1κB下游靶蛋白作用。結(jié)果表明,黃芩素可以顯著的降低TNF-α刺激下,抗凋亡蛋白(cIAP-1、cIAP-2、FLIP、BCL-2)、周期蛋白(Cyclin D1、COX-2)、炎癥因子 IL-8、趨化因子MCP-1、周期相關(guān)蛋白c-Myc、侵襲蛋白MMP-9以及血管形成蛋白VEGF的表達(dá)。通過流式細(xì)胞儀檢測黃芩素對HeLa細(xì)胞凋亡的作用,結(jié)果表明黃芩素能促進(jìn)TNF-α刺激下的細(xì)胞凋亡。通過免疫印跡法證明黃芩素是通過促進(jìn)凋亡相關(guān)蛋白caspase-8和PARP的降解,從而促進(jìn)HeLa細(xì)胞凋亡。通過MTT法證明了黃芩素能顯著抑制HeLa細(xì)胞的增殖。又利用流式細(xì)胞儀檢測黃芩素對細(xì)胞周期的影響。實(shí)驗(yàn)表明,黃芩素使HeLa細(xì)胞停滯在G1期。說明黃芩素抑制HeLa細(xì)胞的增殖是通過抑制細(xì)胞周期進(jìn)程來調(diào)控的。通過免疫印跡法檢測黃芩素對MAPK信號通路的作用。結(jié)果表明,黃芩素能顯著抑制TNF-α刺激下MAPK信號通路中p-p38、p-ERK的表達(dá),而對p-JNK沒有影響。綜上所述,黃芩素可以有效地抑制NF-κB及其下游靶蛋白的表達(dá),進(jìn)而抑制宮頸癌的發(fā)生與發(fā)展。
[Abstract]:Nuclear transcription factor NF- 魏 B is the most common and abundant nuclear transcription factor in mammals. NF- 魏 B can be activated by a variety of stimuli, including genotoxicity, inflammatory factors and oxidative stress. Activation of NF- 魏 B is associated with a variety of cellular processes, including inflammation, transformation, cell proliferation, vascular proliferation, cell invasion, metastasis, chemical resistance and radiation resistance. Therefore, the inhibition of NF- 魏 B by chemotherapeutic drugs can be used as a potential therapy for cancer. Baicalin is a flavonoid compound extracted from the whole grass and rhizome of Chinese medicinal plant Scutellaria baicalensis. Baicalin has been reported to have antioxidant, anti-inflammatory, anti-tumor, anti-anxiety and neuroprotective biological activities. The aim of this study was to study the anti-inflammatory and anti-tumor activity of baicalin targeting NF-1 魏 B and its mechanism. Firstly, the effect of baicalin on NF- 魏 B transcriptional activity was detected by luciferase reporter gene experiment. It was found that baicalein inhibited the transcriptional activity of NF- 魏 B in a concentration-dependent manner under the stimulation of TNF- 偽. MTT assay demonstrated that baicalin had no obvious cytotoxicity to HeLa cells. The effects of baicalin on the expression of I 魏 B 偽, p-I 魏 B 偽 and p65 proteins were detected by Western blot. The results showed that baicalin could inhibit the nuclear transfer of p65 stimulated by TNF- 偽 in HeLa cells by inhibiting the phosphorylation and degradation of I 魏 B 偽 in the cytoplasm. The immunofluorescence assay demonstrated that baicalin could significantly inhibit TNF- 偽 -stimulated p65 entry into the nucleus, thus inhibiting the binding of NF- 魏 B to its promoter. The effect of yellowsyn on the downstream target protein of NF-1 魏 B was detected by Western blot. The results showed that baicalin could significantly reduce cIAP-1,cIAP-2,FLIP,BCL-2, Cyclin D1 COX-2 and IL-8, chemokine MCP-1, stimulated by TNF- 偽. Expression of cyclin c-Myc, invasive protein MMP-9 and angiogenesis protein VEGF. The effect of baicalin on apoptosis of HeLa cells was detected by flow cytometry. The results showed that baicalin could promote apoptosis induced by TNF- 偽. It was proved by Western blotting that baicalin promoted the apoptosis of HeLa cells by promoting the degradation of apoptosis-related proteins caspase-8 and PARP. It was proved by MTT that baicalin could significantly inhibit the proliferation of HeLa cells. The effect of baicalin on cell cycle was also detected by flow cytometry. The results showed that baicalin caused HeLa cells to stagnate in G 1 phase. The results suggest that baicalin inhibits the proliferation of HeLa cells by inhibiting cell cycle progression. The effect of baicalin on MAPK signaling pathway was detected by Western blot. The results showed that baicalin could significantly inhibit the expression of p-p38 p-ERK in MAPK signaling pathway stimulated by TNF- 偽, but had no effect on p-JNK. In conclusion, baicalin can effectively inhibit the expression of NF- 魏 B and its downstream target protein, and then inhibit the occurrence and development of cervical cancer.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285
，

本文編號：2417270