缺氧诱导大鼠肝星状细胞激活及表达内脂素的实验研究

目的 通过观察常氧及缺氧状态下大鼠肝星状细胞(HSCs)表达α-平滑肌肌动蛋白(α-SMA)、缺氧诱导因子-1α（HIF-1α）和内脂素水平的变化，探讨缺氧对HSCs激活及表达内脂素的影响。方法 对原代大鼠HSCs进行缺氧(37 ℃、5% CO2+1% O2 + 94% N2)处理,检测不同缺氧时间(3、6、12及24 h)和常氧条件（5%CO2+21%O2+74%N2）下HSCs中HIF-1α、α-SMA和内脂素的mRNA表达水平及蛋白表达水平。基因表达用RT-PCR检测，蛋白表达用蛋白质印迹法(Western blot) 检测。结果 缺氧3 h即能诱导大鼠HSCs表达HIF-1α mRNA(P<0.05)；HSCs缺氧6 h后α-SMA mRNA及蛋白表达水平的表达上调(P<0.05)；HSCs缺氧12 h上调内脂素mRNA的表达(P<0.05)，6 h上调内脂素蛋白表达(P<0.05)；缺氧诱导α-SMA和内脂素表达呈正相关(基因r=0.991，蛋白r=0.968，P<0.05)。结论 缺氧可上调HSCs表达α-SMA和内脂素，且二者具有相关性，提示缺氧微环境可能通过内脂素介导HSCs的激活，促进肝纤维化的发生。

Effect of Hypoxia on the Activation and Visfatin Expression in Rat Hepatic Stellate Cells

Objective To investigate the effect of hypoxia on the visfatin and the expression of smooth muscle-actin (α-SMA) and hypoxia-inducible factor-1α (HIF-1α) in rat hepatic stellate cells (HSCs). Methods Rat primary HSCs were isolated from SD rats by in situ perfusion of collagenase and pronase and single-step Nycodenz density gradient centrifugation, and then cultured and activated. Completely activated primary HSCs were exposed to hypoxic conditions (37 ℃, 5% CO2, 1% O2,94%N2), or normoxic conditions (37 ℃, 5% CO2, 21% O2,74% N2), for 3, 6, 12 or 24 h respectively. The expression of α-SMA, the marker of HSC activation, and visfatin were assessed by Real time-PCR and Western blot. The Expression of HIF-1α was detected by Real time-PCR. Results HIF-1α mRNA in rat HSCs was induced after exposed to hypoxia for 3 h, and maintained elevated status up to 24 h. HSCs exposed to 1% O2 hypoxic conditions for 6 h increased α-SMA mRNA and protein expression. Visfatin mRNA expression was up-regulated after subjected to hypoxia for 12 h, and protein level was elevated after 6 h hypoxia. A positive linear correlation existed between α-SMA and visfatin expression in responsible to hypoxia 〔r=0.991 (genes) and r=0.968 (proteins),P<0.05〕. Conclusion Microcirculation impairment could significantly induce α-SMA and visfatin expression in rat HSCs, which might potentate the activation process of HSCs.