本文選題：CRYAB + AP-2β��； 參考：《湖南師范大學》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,許多脅迫或病理條件可以誘導CRYAB基因的表達。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胞凋亡,促進細胞存活。在許多神經(jīng)性疾病和惡性腫瘤中CRYAB基因過量表達。因此,研究CRYAB基因的轉(zhuǎn)錄調(diào)控機制具有重要意義。 為了鑒定調(diào)控CRYAB基因轉(zhuǎn)錄的調(diào)節(jié)因子,我們通過生物信息學軟件查找CRYAB基因啟動子上潛在的轉(zhuǎn)錄因子結(jié)合位點。除了以前報道的轉(zhuǎn)錄因子p53結(jié)合位點,我們還發(fā)現(xiàn)了轉(zhuǎn)錄因子AP-2的四個結(jié)合位點。隨后,我們將包含有四個AP-2結(jié)合位點及一個p53結(jié)合位點的CRYAB啟動子序列克隆到熒光素酶報告基因載體pGL3-basic中,并將這個質(zhì)粒分別與AP-2α、AP-2β共轉(zhuǎn)入含野生型p53的細胞系HeLa中,通過熒光素酶分析實驗證明過表達AP-2a或AP-2β都能增強CRYAB啟動子的轉(zhuǎn)錄活性,且AP-2p的作用更強。但在無p53的細胞系H358中,單獨表達AP-2p對CRYAB啟動子活性沒有影響,而共轉(zhuǎn)入AP-2p和p53時CRYAB啟動子活性高于單獨轉(zhuǎn)入p53時的活性。這些結(jié)果暗示AP-2β通過p53增強CRYAB啟動子活性。為了進一步驗證這個結(jié)果,我們利用了一個只含有p53結(jié)合位點而沒有AP-2結(jié)合位點的熒光素酶報告基因載體pp53-TA-Luc去檢測p53轉(zhuǎn)錄激活活性。將pp53-TA-Luc與AP-2p、p53共轉(zhuǎn)入H358細胞中,發(fā)現(xiàn)單獨轉(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,pp53-TA-Luc活性比單獨轉(zhuǎn)染p53時提高60.4%,說明AP-2β能增強p53的轉(zhuǎn)錄激活活性。 為了探討AP-2p增強p53的轉(zhuǎn)錄激活活性的分子機制,我們通過免疫共沉淀實驗證實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共表達時,AP-2β能增強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p53的穩(wěn)定性,從而增強由p53介導的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.
【學位授予單位】：湖南師范大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R346

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