Zhu Yong-Jun, Hu Xiao-Lin, Men Ruo-Ting, Yang Li
Department of Gastroenterology, West China Hospital, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2013 May;44(3):352-6.
To investigate the role of macrophage migration inhibitory factor (MIF) in activation of primary hepatic stellate cells (HSC), and to explore the blocking effect of specific antisense oligonucleotides (ASONs) targeting MIF on the activation of primary HSCs.
Rat primary HSC 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. The expression of alpha-smooth muscle actin (alpha-SMA), the marker of HSC activation, was assessed by RT-PCR and immunocytochemistry (ICC). The expression of MIF was detected by RT-PCR, enzyme-linked immuno sorbent assay (ELISA), immunofluorescence (IF) or ICC. Completely activated primary HSC were transfected with MIF-specific antisense oligonucleotide (MIF-ASONs), mis-sense oligonucleotides of MIF, or blank liposome (negative-control).
Quiescent HSC expressed very low level of MIF, while MIF gene and protein expression increased significantly during the process of HSC activation. Positive correlation was found between the expression of alpha-SMA and MIF (r = 0.944, P < 0.01). Compared with the control, activated primary Gen HSC transfected with MIF-ASONs showed lower MIF and alpha-SMA expressions at gene and protein profiles (P < 0.05).
The expression of MIF was up-regulated in the process of HSC activation. Activation in culture of primarily isolated rat HSC could be inhibited by MIF-ASONs, suggesting at least in part that, MIF might be a novel and important factor involved in HSC activation and hepatic fibrosis.
研究巨噬细胞移动抑制因子（MIF）在原代肝星状细胞（HSC）激活中的作用，并探讨靶向MIF的特异性反义寡核苷酸（ASONs）对原代HSCs激活的阻断作用。
采用胶原酶和链霉蛋白酶原位灌注及一步法Nycodenz密度梯度离心从SD大鼠分离原代HSC，然后进行培养和激活。通过RT-PCR和免疫细胞化学（ICC）评估HSC激活标志物α-平滑肌肌动蛋白（α-SMA）的表达。通过RT-PCR、酶联免疫吸附测定（ELISA）、免疫荧光（IF）或ICC检测MIF的表达。用MIF特异性反义寡核苷酸（MIF-ASONs）、MIF错义寡核苷酸或空白脂质体（阴性对照）转染完全激活的原代HSC。
静止HSC表达极低水平的MIF，而在HSC激活过程中MIF基因和蛋白表达显著增加。发现α-SMA和MIF的表达之间呈正相关（r = 0.944，P < 0.01）。与对照组相比，用MIF-ASONs转染的激活原代Gen HSC在基因和蛋白水平上显示出较低的MIF和α-SMA表达（P < 0.05）。
HSC激活过程中MIF表达上调。MIF-ASONs可抑制原代分离大鼠HSC的培养激活，这至少部分表明，MIF可能是参与HSC激活和肝纤维化的一个新的重要因子。
