【摘要】：核轉(zhuǎn)錄因子進(jìn)入細(xì)胞核是其發(fā)揮轉(zhuǎn)錄活性的前提。研究發(fā)現(xiàn)信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄活性因子(Signal transducers and activators of transcription, STATs)家族成員不具有引導(dǎo)蛋白入核的核定位序列(nuclear location signal, NLS),但是細(xì)胞因子的刺激可以導(dǎo)致STAT短暫胞核聚集現(xiàn)象,因此研究STAT入核機(jī)制是一個(gè)值得探討的課題。研究發(fā)現(xiàn),STAT1和STAT3的DNA結(jié)合域中一段序列具有促進(jìn)其進(jìn)入細(xì)胞核的功能。那么STAT4的DNA結(jié)合域中是否也存在類似的序列,能否引導(dǎo)STAT4入核,目前尚無(wú)報(bào)道。 目的：探討STAT4受IL-12刺激后移位入細(xì)胞核的機(jī)制。 方法： 1.采用生物信息學(xué)方法將STAT1和STAT3的DNA結(jié)合域與STAT4的DNA結(jié)合域進(jìn)行比對(duì),以期找到STAT4DNA結(jié)合域上的同源序列。 2.以pEGFP-C1為表達(dá)載體、采用基因亞克隆的方法構(gòu)建質(zhì)粒,包括：缺失395-416位氨基酸殘基序列的缺失型STAT4質(zhì)粒即pEGFP-STAT4-Del、將SV40大T抗原上經(jīng)典的NLS核酸序列插入表達(dá)載體的陽(yáng)性對(duì)照質(zhì)粒pEGFP-NLS、將NLS插入缺失型STAT4的pEGFP-NLS-STAT4-Del質(zhì)粒,這些質(zhì)粒經(jīng)過(guò)酶切消化鑒定和DNA測(cè)序鑒定,并采用免疫印跡方法確定這些質(zhì)粒在真核細(xì)胞中表達(dá) 3.將上述質(zhì)粒轉(zhuǎn)染Caski細(xì)胞,通過(guò)IL-12刺激,熒光顯微鏡觀察熒光信號(hào)的亞細(xì)胞定位。結(jié)果： 1.經(jīng)過(guò)生物信息學(xué)分析發(fā)現(xiàn)STAT4DNA結(jié)合域上395-416位氨基酸殘基序列可能是STAT4潛在的二聚體特異性NLS (dimer-specific NLS, dsNLS)。 2.經(jīng)過(guò)酶切鑒定和DNA序列測(cè)定證明構(gòu)建了正確的質(zhì)粒,包括：pEGFP-STAT4、pEGFP-STAT4-Del、pEGFP-NLS和pEGFP-NLS-STAT4-Del,將這些質(zhì)粒轉(zhuǎn)染真核細(xì)胞都能正確表達(dá)。 3.將pEGFP-STAT4、pEGFP-STAT4-Del分別轉(zhuǎn)染Caski細(xì)胞,IL-12刺激后發(fā)現(xiàn)野生型STAT4進(jìn)入細(xì)胞核,而缺失型STAT4在IL-12刺激前后,都主要分布在細(xì)胞漿。進(jìn)一步用leptomycin B (LMB)處理轉(zhuǎn)染細(xì)胞,再用IL-12刺激,發(fā)現(xiàn)刺激Omin時(shí)野生型STAT4分布在胞漿,45min后進(jìn)入細(xì)胞核,60min后陽(yáng)性信號(hào)仍然在細(xì)胞核里,表明野生型STAT4受工L-12刺激被活化,形成同源二聚體,以這種形式移入細(xì)胞核；但缺失型STAT4轉(zhuǎn)染細(xì)胞用LMB處理后,再用工L-12刺激,大部分陽(yáng)性信號(hào)始終分布在細(xì)胞漿,表明缺失型STAT4確實(shí)不能進(jìn)入細(xì)胞核；將經(jīng)典的NLS序列插入缺失型STAT4中,轉(zhuǎn)染該質(zhì)粒,發(fā)現(xiàn)含有NLS的缺失型STAT4也能進(jìn)入細(xì)胞核。表明STAT4的395-416位氨基酸殘基序列在引導(dǎo)STAT4入核中起重要作用。 結(jié)論： STAT4的395-416位氨基酸序列具有dsNLS功能,在IL-12刺激下,能夠引導(dǎo)STAT4入核。
[Abstract]:The entry of nuclear transcription factors into the nucleus is a prerequisite for their transcriptional activity. It was found that members of the (Signal transducers and activators of transcription, STATs) family of signal transduction and transcriptional activity factors do not have a nuclear localization sequence (nuclear location signal, NLS), that leads to the entry of proteins into the nucleus. However, cytokine stimulation can lead to the transient nuclear aggregation of STAT. Therefore, the study of STAT nucleation mechanism is a worthy topic. It was found that a sequence in the DNA binding domain of STAT1 and STAT3 promoted their entry into the nucleus. So whether there are similar sequences in the DNA binding domain of STAT4 and whether the STAT4 can be guided into the core is not reported. Objective: to investigate the mechanism of STAT4 translocation into nucleus after IL-12 stimulation. Methods: 1. The DNA binding domain of STAT1 and STAT3 was compared with the DNA binding domain of STAT4 by bioinformatics in order to find the homologous sequences on STAT4DNA binding domain. 2. Using pEGFP-C1 as expression vector, the plasmid was constructed by gene subcloning method, including: deletion type STAT4 plasmid (pEGFP-STAT4-Del,) with deletion of 395-416 amino acid residues. The classical NLS nucleic acid sequence on the large T antigen of SV40 was inserted into the positive control plasmid pEGFP-NLS, of the expression vector, and the NLS was inserted into the pEGFP-NLS-STAT4-Del plasmid of the deletion type STAT4. These plasmids were digested and identified by restriction endonuclease digestion and DNA sequencing. The expression of these plasmids in eukaryotic cells was determined by Western blot. The above plasmids were transfected into Caski cells. The subcellular localization of fluorescent signals was observed by IL-12 stimulation and fluorescence microscope. Results: 1. Bioinformatics analysis revealed that the amino acid residues at 395-416 on STAT4DNA binding domain may be the potential dimer specific NLS (dimer-specific NLS, dsNLS).) of STAT4. 2. The results of restriction endonuclease digestion and DNA sequencing showed that the correct plasmids were constructed, including pEGFP-STAT4,pEGFP-STAT4-Del,pEGFP-NLS and pEGFP-NLS-STAT4-Del, transfection of these plasmids into eukaryotic cells. 3. After pEGFP-STAT4,pEGFP-STAT4-Del was transfected into Caski cells, the wild type STAT4 was found to enter the nucleus after IL-12 stimulation, while the deletion type STAT4 was mainly distributed in the cytoplasm before and after IL-12 stimulation. After treated with leptomycin B (LMB) and stimulated by IL-12, it was found that the wild-type STAT4 was distributed in the cytoplasm of Omin, entered into the nucleus after 45min, and the positive signal of 60min was still in the nucleus. The results showed that wild type STAT4 was activated by L-12 to form homologous dimer and transferred into nucleus in this form. However, after treated with LMB, most of the positive signals were distributed in the cytoplasm of the deleted STAT4 transfected cells, which indicated that the deletion type STAT4 could not enter the nucleus. The classical NLS sequence was inserted into the deletion type STAT4 and transfected into the plasmid. It was found that the deletion type STAT4 containing NLS could also enter the nucleus. These results suggest that the amino acid residues at position 395-416 of STAT4 play an important role in guiding STAT4 into the nucleus. Conclusion: the amino acid sequence at position 395-416 of STAT4 has the function of dsNLS and can induce STAT4 into nucleus under the stimulation of IL-12.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392.1

【共引文獻(xiàn)】

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