【摘要】：目前的研究認(rèn)為,NF-κB介導(dǎo)的信號(hào)通路可廣泛參與細(xì)胞存活、發(fā)育和腫瘤進(jìn)展等過(guò)程。盡管決定NF-κB從細(xì)胞漿到細(xì)胞核轉(zhuǎn)位的分子機(jī)制已被廣泛報(bào)道,對(duì)于細(xì)胞核內(nèi)NF-κB活性的調(diào)控機(jī)制尚不十分清楚。既往研究表明,LRP16作為macro domain蛋白家族的一個(gè)特殊成員,屬于雌激素受體和雄激素受體的共激活因子；與NF-κB的共激活因子UXT存在相互作用。在本課題中,我們主要研究了LRP16對(duì)NF-κB活性的調(diào)控作用。方法：首先,通過(guò)GST pull -down和免疫共沉淀實(shí)驗(yàn)驗(yàn)證LRP16與p65之間的相互作用。然后通過(guò)熒光素酶活性分析、電泳遷移率檢測(cè)、Real-time PCR、染色質(zhì)免疫共沉淀等方法檢測(cè)了LRP16對(duì)NF-κB的報(bào)告基因活性、NF-κB與靶DNA的結(jié)合能力、NF-кB下游靶基因的表達(dá)水平及NF-κB與靶基因啟動(dòng)子區(qū)域結(jié)合能力的影響。并通過(guò)Annexin V標(biāo)記與流式細(xì)胞計(jì)數(shù)的方法檢測(cè)了LRP16對(duì)TNF-α誘導(dǎo)細(xì)胞凋亡的影響。最終通過(guò)免疫組化染色和酶聯(lián)免疫吸附實(shí)驗(yàn)研究了人類原位胃癌樣本中胞核分布的LRP16對(duì)NF-κB活性的影響。結(jié)果：LRP16通過(guò)與p65相互作用參與NF-κB轉(zhuǎn)錄復(fù)合體的形成;通過(guò)RNA干擾技術(shù)沉默細(xì)胞中內(nèi)源性LRP16的表達(dá)可以降低NF-кB活性并明顯下調(diào)NF-κB下游靶基因的表達(dá)。機(jī)制研究顯示沉默LRP16表達(dá)并不影響TNF-α誘導(dǎo)的NF-κB核轉(zhuǎn)位,但能影響細(xì)胞核中NF-KB/p300/CREB功能轉(zhuǎn)錄復(fù)合體的形成和穩(wěn)定。此外,干擾LRP16的表達(dá)后也能增加細(xì)胞對(duì)TNF-α誘導(dǎo)凋亡的敏感性。最后,在人類原位胃癌標(biāo)本中初步確立核內(nèi)LRP16表達(dá)陰性組的NF-κB活性整體低于LRP16表達(dá)陽(yáng)性組的NF-κB活性。結(jié)論：本研究表明LRP16不僅是細(xì)胞核內(nèi)NF-κB;舌性的一個(gè)關(guān)鍵調(diào)控因子,對(duì)腫瘤中NF-κB的異常激活也有很重要的作用。
[Abstract]:It has been suggested that NF- 魏 B mediated signaling pathway is involved in cell survival, development and tumor progression. Although the molecular mechanism that determines the translocation of NF- 魏 B from cytoplasm to nucleus has been widely reported, the regulatory mechanism of NF- 魏 B activity in the nucleus is not well understood. Previous studies have shown that LRP16, as a special member of the macro domain protein family, is a coactivator of estrogen receptor and androgen receptor, and interacts with NF- 魏 B coactivator UXT. In this study, we studied the regulation of NF- 魏 B activity by LRP16. Methods: firstly, the interaction between LRP16 and p65 was verified by GST pull-down and immunoprecipitation. Then the reporter gene activity of LRP16 to NF- 魏 B and the binding ability of NF- 魏 B to target DNA were detected by luciferase activity analysis, electrophoretic mobility assay and Real-time PCR, chromatin immunoprecipitation. The expression level of target gene downstream of NF- B and the binding ability of NF- 魏 B to the promoter region of target gene. The effect of LRP16 on apoptosis induced by TNF- 偽 was detected by Annexin V labeling and flow cytometry. Finally, the effect of nuclear distribution of LRP16 on the activity of NF- 魏 B was studied by immunohistochemical staining and enzyme-linked immunosorbent assay (Elisa). Results: LRP16 was involved in the formation of NF- 魏 B transcriptional complex by interacting with p65, and the expression of endogenous LRP16 in cells was inhibited by RNA interference technique, and the expression of downstream target gene of NF- 魏 B was significantly down-regulated by RNA interference. The mechanism study showed that the silencing of LRP16 expression did not affect the nuclear translocation of NF- 魏 B induced by TNF- 偽, but affected the formation and stability of NF-KB/p300/CREB functional transcriptional complex in the nucleus. In addition, interfering with the expression of LRP16 increased the sensitivity of cells to apoptosis induced by TNF- 偽. Finally, it was preliminarily established that the activity of NF- 魏 B in nuclear LRP16 negative group was lower than that in LRP16 positive group. Conclusion: LRP16 is not only a key regulatory factor of NF- 魏 B in the nucleus, but also plays an important role in the abnormal activation of NF- 魏 B in tumor.
【學(xué)位授予單位】：中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 陳錦汝;冠心合劑主要活性成分對(duì)TNF-α損傷人臍靜脈內(nèi)皮細(xì)胞的保護(hù)作用及其機(jī)制的研究[D];浙江中醫(yī)藥大學(xué);2013年

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本文編號(hào)：2367466