本文選題：CRYAB + AP-2β��； 參考：《湖南師范大學(xué)》2011年碩士論文

【摘要】：晶狀體蛋白CRYAB是小熱激蛋白家族的一員,許多脅迫或病理?xiàng)l件可以誘導(dǎo)CRYAB基因的表達(dá)。在脅迫條件下CRYAB作為分子伴侶起作用,可阻止變性蛋白聚集和細(xì)胞凋亡,促進(jìn)細(xì)胞存活。在許多神經(jīng)性疾病和惡性腫瘤中CRYAB基因過(guò)量表達(dá)。因此,研究CRYAB基因的轉(zhuǎn)錄調(diào)控機(jī)制具有重要意義。 為了鑒定調(diào)控CRYAB基因轉(zhuǎn)錄的調(diào)節(jié)因子,我們通過(guò)生物信息學(xué)軟件查找CRYAB基因啟動(dòng)子上潛在的轉(zhuǎn)錄因子結(jié)合位點(diǎn)。除了以前報(bào)道的轉(zhuǎn)錄因子p53結(jié)合位點(diǎn),我們還發(fā)現(xiàn)了轉(zhuǎn)錄因子AP-2的四個(gè)結(jié)合位點(diǎn)。隨后,我們將包含有四個(gè)AP-2結(jié)合位點(diǎn)及一個(gè)p53結(jié)合位點(diǎn)的CRYAB啟動(dòng)子序列克隆到熒光素酶報(bào)告基因載體pGL3-basic中,并將這個(gè)質(zhì)粒分別與AP-2α、AP-2β共轉(zhuǎn)入含野生型p53的細(xì)胞系HeLa中,通過(guò)熒光素酶分析實(shí)驗(yàn)證明過(guò)表達(dá)AP-2a或AP-2β都能增強(qiáng)CRYAB啟動(dòng)子的轉(zhuǎn)錄活性,且AP-2p的作用更強(qiáng)。但在無(wú)p53的細(xì)胞系H358中,單獨(dú)表達(dá)AP-2p對(duì)CRYAB啟動(dòng)子活性沒(méi)有影響,而共轉(zhuǎn)入AP-2p和p53時(shí)CRYAB啟動(dòng)子活性高于單獨(dú)轉(zhuǎn)入p53時(shí)的活性。這些結(jié)果暗示AP-2β通過(guò)p53增強(qiáng)CRYAB啟動(dòng)子活性。為了進(jìn)一步驗(yàn)證這個(gè)結(jié)果,我們利用了一個(gè)只含有p53結(jié)合位點(diǎn)而沒(méi)有AP-2結(jié)合位點(diǎn)的熒光素酶報(bào)告基因載體pp53-TA-Luc去檢測(cè)p53轉(zhuǎn)錄激活活性。將pp53-TA-Luc與AP-2p、p53共轉(zhuǎn)入H358細(xì)胞中,發(fā)現(xiàn)單獨(dú)轉(zhuǎn)染AP-2β不影響pp53-TA-Luc熒光素酶活性,而AP-2p和p53共轉(zhuǎn)染時(shí),pp53-TA-Luc活性比單獨(dú)轉(zhuǎn)染p53時(shí)提高60.4%,說(shuō)明AP-2β能增強(qiáng)p53的轉(zhuǎn)錄激活活性。 為了探討AP-2p增強(qiáng)p53的轉(zhuǎn)錄激活活性的分子機(jī)制,我們通過(guò)免疫共沉淀實(shí)驗(yàn)證實(shí)AP-2p蛋白能和p53蛋白相互作用。并且,我們還發(fā)現(xiàn)AP-2β和p53共表達(dá)時(shí),AP-2β能增強(qiáng)p53蛋白的穩(wěn)定性。由此我們得到結(jié)論,AP-2β與p53結(jié)合并加強(qiáng)p53的穩(wěn)定性,從而增強(qiáng)由p53介導(dǎo)的CRYAB基因的轉(zhuǎn)錄。
[Abstract]:Crystallin CRYAB is a member of small heat shock protein family. Many stress or pathological conditions can induce the expression of CRYAB gene. Under stress, CRYAB, acting as a molecular chaperone, can prevent denatured protein aggregation and cell apoptosis and promote cell survival. CRYAB gene is overexpressed in many neurological diseases and malignant tumors. Therefore, it is of great significance to study the transcriptional regulation mechanism of CRYAB gene. In order to identify the regulatory factors that regulate the transcription of CRYAB gene, we searched the potential transcription factor binding sites on the promoter of CRYAB gene by bioinformatics software. In addition to previously reported transcription factor p53 binding sites, we also found four binding sites of transcription factor AP-2. Subsequently, we cloned the CRYAB promoter sequence containing four AP-2 binding sites and one p53 binding site into the luciferase reporter gene vector pGL3-basic, and cotransfected the plasmid with AP-2 偽 AP-2 尾 into the wild type p53 cell line HeLa. The results of luciferase analysis showed that overexpression of AP-2a or AP-2 尾 could enhance the transcriptional activity of CRYAB promoter, and AP-2p played a more important role. However, in the cell line H358 without p53, the expression of AP-2p alone had no effect on the activity of CRYAB promoter, but the activity of CRYAB promoter was higher in co-transfer of AP-2p and p53 than that in p53 alone. These results suggest that AP-2 尾 enhances the activity of CRYAB promoter through p53. To further verify this result, we used pp53-TA-Luc, a luciferase reporter gene vector containing only p53 binding sites but no AP-2 binding sites, to detect p53 transcriptional activation. Pp53-TA-Luc and AP-2ptp53 were co-transfected into H358 cells. It was found that transfection of AP-2 尾 alone did not affect the activity of pp53-TA-Luc luciferase, while the activity of pp53-TA-Luc luciferase in AP-2p and p53 co-transfection was 60.4% higher than that in p53 alone, indicating that AP-2 尾 could enhance the transcriptional activation of p53. In order to investigate the molecular mechanism of AP-2p enhancing p53 transcriptional activation, we demonstrated that AP-2p protein can interact with p53 protein by immunoprecipitation. Moreover, we also found that AP-2 尾 can enhance the stability of p53 protein when AP-2 尾 and p53 co-express. It is concluded that AP-2 尾 binds to p53 and enhances the stability of p53, thus enhancing the transcription of CRYAB gene mediated by p53.
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346

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