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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 ,在其转录调控中起重要作用
Objective To investigate the role of live tissue activator of transcription (LAP), a hepatic tissue-specific transcription factor, in the regulation of α1 (I) gene expression in activated liver stellate cells (HSCs). Methods The rat HSCs were isolated by pronase injection of streptavidin, collagenase and Nycodenz density gradient centrifugation, and then cultured in vitro to activate them. A luciferase reporter plasmid containing the human α1 (I) collagen gene promoter fragment (-80 4 to +14 5 2 or -80 4 to +2 2 2 bases) was constructed. The constructed reporter plasmids were transiently co-transfected into activated HSC cells with LAP expression plasmids using cationic liposome-mediated methods. The activity of the α1 (I) gene promoter was determined by measuring luciferase activity. As a result, the luciferase reporter gene (PGL3 col) containing the first intron fragment of the α1 (I) gene was constructed as compared with the reporter gene PGL3 col (Δintron) without the first intron of the α1 (I) gene HSC was highly expressed (luciferase activity was 3 15 ± 45 U / mg protein and 220 ± 70 U / mg protein, P <0.05). Transient co-transfection of LAP expression plasmids significantly enhanced the expression of PGL3 col and PGL3 col (Δintron) reporter genes in HSCs with luciferase activity of 5 87 ± 62 U / mg protein and 3 15 ± 45 U / mg protein and 3 2 6 ± 5 2 U / mg protein and 220 ± 70 U / mg protein, both P <0.05). Conclusion LAP can transactivate (induce) α1 (I) gene in activated HSC and play an important role in its transcriptional regulation