摘要
目的 了解激活的肝脏星状细胞 (hepaticstellatecell,HSC)中一种肝脏组织特异的转录因子 肝脏激活蛋白 (liveractivatorprotein ,LAP)在α1(I)基因表达调控中的作用。方法 采用链霉蛋白酶、胶原酶原位灌注、Nycodenz密度梯度离心分离大鼠HSC ,并进行体外培养使之活化。构建含人α1(I)胶原基因启动子片段 ( -80 4～ +14 5 2或 -80 4～ +2 2 2碱基 )的荧光素酶报告基因质粒。用构建的报告基因质粒与LAP表达质粒一起 ,用阳离子脂质体介导的方法 ,瞬时共转染激活的HSC细胞。α1(I)基因启动子的活性通过测定荧光素酶活性来确定。结果 构建的含α1(I)基因第一内含子片段的荧光素酶报告基因 (PGL3 col)比不含α1(I)基因第一内含子的报告基因〔PGL3 col( △intron)〕在HSC中有较强的表达 (荧光素酶活性为 3 15± 45U/mg蛋白与 2 2 0± 70U /mg蛋白 ,P <0 .0 5 )。瞬时共转染LAP表达质粒可明显增强PGL3 col及PGL3 col( △intron)报告基因在HSC中的表达 (荧光素酶活性分别为 5 87± 62U /mg蛋白与3 15± 45U /mg蛋白及 3 2 6± 5 2U/mg蛋白与 2 2 0± 70U /mg蛋白 ,两者比较 ,P <0 .0 5 )。 结论 LAP可以反式激活 (诱导 )α1(I)基因在活化的HSC中表达 。
Objective To elucidate the role of the transcription factor liver activator protein (LAP, a member of the C/EBP family) in the expression of α 1(I) collagen gene in activated hepatic stellate cells (HSCs). Methods Rat HSCs were prepared from SD rats by in situ perfusion and single step density Nycodenz gradient. Two chimeric luciferase reporter gene plasmids containing the human collagen α 1(I) gene promoter fragments (-804～+1 452 or -804～+222) were constructed. Culture activated HSCs were co transfected with the reporter gene contructs and mammalian vector expressing LAP using the cationic liposome mediated method, and the promoter activity was determined by measuring luciferase activity. Results The luciferase reporter gene construct containing the first intron of α 1(I) collagen gene (-804～+1 452, was called as PGL3 col) had a higher level of gene expression, as compared with the construct lacking the first intron 〔was called as PGL3 col (△intron)〕 in activated HSCs (315±45 U/mg protein vs 220±70 U/mg protein, P <0.05). Transient transfection of the vector expressing LAP significantly increased basal transcription from PGL3 col and PGL3 col (△intron) reporter gene vectors (587±62 U/mg protein vs 315±45 U/mg protein and 326±52 U/mg protein vs 220±70 U/mg protein respectively, both P < 0.05). Conclusion The transcription factor LAP transactivates collagen α 1(I) gene in activated HSCs, and the first intron is important for α 1(I) collagen gene transcription activity in activated HSCs.
出处
《中国普外基础与临床杂志》
CAS
2000年第2期79-82,共4页
Chinese Journal of Bases and Clinics In General Surgery
关键词
转录因子
肝脏激活蛋白
基因调控
肝脏星状细胞
Transcription factor Liver activator protein α _1(I) collagen gene regulation Hepatic stellate cell
