黄芩素对宫颈癌细胞中NF-kB及其下游靶基因表达的影响
发布时间：2019-01-28 20:00
【摘要】：在哺乳动物中,核转录因子NF-κB是一类最常见、含量最丰富的核转录因子,NF-κB可被多种刺激因素激活,包括:基因毒性、炎症因子以及氧化应激。在肿瘤的发生发展中,活化的NF-κB与多种细胞过程相关,包括:炎症、转化、细胞增殖、血管增生、细胞侵袭、转移以及化学抵抗和抗辐射性。因此,化疗药物对NF-κB的抑制可作为治疗肿瘤的潜在治疗手段。黄芩素是从中国药用植物黄芩的全草、根茎中提取出来的黄酮类化合物。已报道黄芩素具有抗氧化、抗炎、抗肿瘤、抗焦虑以及神经保护等生物活性。本实验主要针对黄芩素以NF-1κB为靶点的抗炎抗肿瘤活性及其作用机制进行研究。首先,通过荧光素酶报告基因实验检测黄芩素对NF-κB转录活性的作用。发现在TNF-α刺激下,黄芩素可浓度依赖型抑制NF-κB转录活性。MTT实验证明了黄芩素对HeLa细胞没有明显的细胞毒性。通过免疫印迹法检测了黄芩素对IκBα、p-IκBα、p65蛋白表达的作用。结果发现,黄芩素可以通过抑制细胞质内IκBα的磷酸化与降解来抑制HeLa细胞中TNF-α刺激下p65的核转移。通过免疫荧光法再次验证了黄芩素能显著抑制TNF-α刺激下P65进入细胞核,从而抑制了 NF-κB与其启动子的结合。通过免疫印迹法检测黄岑素对NF-1κB下游靶蛋白作用。结果表明,黄芩素可以显著的降低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)、炎症因子 IL-8、趋化因子MCP-1、周期相关蛋白c-Myc、侵袭蛋白MMP-9以及血管形成蛋白VEGF的表达。通过流式细胞仪检测黄芩素对HeLa细胞凋亡的作用,结果表明黄芩素能促进TNF-α刺激下的细胞凋亡。通过免疫印迹法证明黄芩素是通过促进凋亡相关蛋白caspase-8和PARP的降解,从而促进HeLa细胞凋亡。通过MTT法证明了黄芩素能显著抑制HeLa细胞的增殖。又利用流式细胞仪检测黄芩素对细胞周期的影响。实验表明,黄芩素使HeLa细胞停滞在G1期。说明黄芩素抑制HeLa细胞的增殖是通过抑制细胞周期进程来调控的。通过免疫印迹法检测黄芩素对MAPK信号通路的作用。结果表明,黄芩素能显著抑制TNF-α刺激下MAPK信号通路中p-p38、p-ERK的表达,而对p-JNK没有影响。综上所述,黄芩素可以有效地抑制NF-κB及其下游靶蛋白的表达,进而抑制宫颈癌的发生与发展。
[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- 伪. MTT assay demonstrated that baicalin had no obvious cytotoxicity to HeLa cells. The effects of baicalin on the expression of I 魏 B 伪, p-I 魏 B 伪 and p65 proteins were detected by Western blot. The results showed that baicalin could inhibit the nuclear transfer of p65 stimulated by TNF- 伪 in HeLa cells by inhibiting the phosphorylation and degradation of I 魏 B 伪 in the cytoplasm. The immunofluorescence assay demonstrated that baicalin could significantly inhibit TNF- 伪 -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- 伪. 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- 伪. 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- 伪, 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.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285
，
本文编号：2417270
[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- 伪. MTT assay demonstrated that baicalin had no obvious cytotoxicity to HeLa cells. The effects of baicalin on the expression of I 魏 B 伪, p-I 魏 B 伪 and p65 proteins were detected by Western blot. The results showed that baicalin could inhibit the nuclear transfer of p65 stimulated by TNF- 伪 in HeLa cells by inhibiting the phosphorylation and degradation of I 魏 B 伪 in the cytoplasm. The immunofluorescence assay demonstrated that baicalin could significantly inhibit TNF- 伪 -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- 伪. 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- 伪. 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- 伪, 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.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285
，
本文编号：2417270
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