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目的:研究核仁素的转录激活子作用,探讨CD_(34)~+分子的基因转录调节机制。方法:分别构建核仁素的表达质粒,以及野生型和含有缺失突变的CD_(34)~+基因调控区质粒,启动子区下游含有荧光素酶报告基因。这些报告质粒的调控区分别是:①0.8kb野生型CD_(34)~+基因上游5’端序列;②缺失了127个核苷酸的CD_(34)~+基因上游5’端序列。应用瞬时转染试验,研究人CD_(34)~+基因上游5’端的转录特征,分析核仁素对CD_(34)~+基因的转录调节功能。结果:①含野生型CD_(34)~+基因上游5’端序列的报告质粒的相对荧光素酶活性约为空质粒pGL3-Basic的3倍;CD_(34)~+基因上游调控区的活性可被核仁素激活,其相对荧光素酶活性是空质粒的8倍。②CD_(34)~+基因上游5’端有127nt.缺失的报告质粒的相对荧光素酶活性与空质粒pGL3-Basic的活性相同,且与核仁素表达质粒共转染细胞时,其相对荧光素酶活性没有变化。结论:核仁素对CD_(34)~+基因起转录激活作用。CD_(34)~+基因上游5’端自-470nt.至-344nt.的区域对CD_(34)~+基因的表达具有重要意义。
OBJECTIVE: To study the transcriptional activator of nucleolin, and to explore the mechanism of gene transcription regulation of CD_ (34) ~ +. Methods: The expression plasmids of nucleolin were constructed respectively, and the wild-type and the deletion-mutated CD_ (34) ~ + gene regulatory plasmids were constructed. The luciferase reporter gene was located downstream of the promoter. The regulatory regions of these reporter plasmids are: ①0.8kb 5’-upstream sequence of wild-type CD_ (34) ~ + gene; ② 5’-terminal sequence of CD_ (34) ~ + gene with 127 nucleotides deleted. Transient transfection experiments were performed to study the transcriptional features of human CD_ (34) ~ + upstream 5 ’end and the transcriptional regulation of CD_ (34) ~ + by nucleolin. Results: ① The relative luciferase activity of the reporter plasmids containing wild type CD_ (34) ~ + upstream 5 ’end was about 3 times that of the empty plasmid pGL3-Basic; the activity of the upstream regulatory region of CD_ (34) ~ + Can be activated by nucleolin, its relative luciferase activity is eight times the empty plasmid. ② The relative luciferase activity of the deleted reporter plasmids was the same as that of the empty plasmid pGL3-Basic, and when compared with the nucleolin expression plasmid, the relative fluorescence No change in enzyme activity. Conclusion: Nucleolin can activate transcription of CD34 + gene. The upstream region of CD34 + gene from -470 nt to -344 nt is of great importance for the expression of CD34 +.