COX-2抑制HSC细胞凋亡在肝纤维化形成中具有重要地位，但COX-2抑制HSC细胞凋亡的机制不清，我们前期研究及预实验发现COX-2对HSC细胞Acsl6表达及神经酰胺含量具有下调作用，因此提出COX-2可能通过下调Acsl6-神经酰胺通路抑制HSC凋亡参与肝纤维化的假说。本项目将在动物水平上首先明确纤维化肝组织具有COX-2上调、Acsl6-神经酰胺通路下调、HSC凋亡下降和显著增殖的特征。然后在细胞水平上明确高表达COX-2基因可下调HSC细胞Acsl6-神经酰胺通路和抑制其凋亡，而沉默COX-2基因可上调HSC细胞Acsl6-神经酰胺通路和促进其凋亡；阐明阻断Acsl6-神经酰胺通路不仅可促进HSC增殖、抑制其凋亡，而且可逆转沉默COX-2基因的促HSC凋亡作用，从Acsl6-神经酰胺通路这一新视角揭示COX-2抑制HSC凋亡机制，为深入研究肝纤维化发病机制及其防治提供新的思路。

COX-2-inhibited apoptosis of hepatic stellate cells （HSC） plays an important role in the formation of liver fibrosis,but the mechanism underlying COX-2-inhibited HSC apoptosis is unclear.Our previous studies and preliminary experiments found that the expression of Acsl6 and content of ceramide were down-regulated by COX-2 in the activated hepatic stellate cells.Therefore,we propose a working hypothesis that COX-2 is invovled in the formation of hepatic fibrosis by inhibiting aopotosis of hepatic stellate cells through down-regulation of Acsl6-ceramide pathway.We will first to prove that the liver tissues of fibrosis have the features of COX-2 up-regulation,Acsl6-ceramide pathway down-regulation, apoptosis decrease and proliferation increase in the animal model of liver fibrosis. And then,We are to clarify at the cellular level that overexpression of COX-2 gene may down-regulate the Acsl6-ceramide pathway and inhibit the HSC apoptosis,and that the silence of COX-2 gene may up-regulate Acsl6-ceramide pathway and induces the HSC apoptosis,and that inhibiting the Acsl6-ceramide pathway not only promotes HSC proliferation and inhibits HSC apoptosis but also reverses the silence of COX-2 gene-promoted HSC apoptosis.This work is to reveal the mechanism of COX-2-inhibited HSC apoptosis and provide new ideas for deeply studying the pathogenesis of liver fibrosis and its prevention and therapies from the new perspective of Acsl6-ceramide pathway.

环氧合酶-2 (Cyclooxgenase-2, COX-2)作为一种诱导型酶，在正常情况下，人类和大鼠肝细胞几乎不表达，但在慢性肝炎和肝硬化的肝组织中呈高表达，其表达量随着肝纤维化程度的加重而增加，提示COX-2参与肝纤维发生发展过程。但COX-2参与肝纤维化发生发展机制不清。本研究应用腺病毒介导的重组干扰质粒（COX-2shRNA）肝纤维化模型大鼠尾静脉注射，肝纤维化程度评分及胶原面积分析发现，除正常对照组（A）外,其余各组大鼠肝纤维化不同程度增加（B= 模型组,C=空载体组,D=COX-2shRNA组），胶原面积不同程度增大（ABCD组分别为0.63 ± 0.24；9.65±1.11；9.34± 1.02；3.08± 0.55），差异具有显著性（P<0.01）。其中COX-2shRNA治疗组纤维化较模型组及空载体组轻。COX-2shRNA沉默COX-2基因抑制肝纤维化的作用机制与下列因素有关：（1）抑制肝星状细胞增殖/诱导肝星状细胞凋亡。肝组织a-SMA阳性面积（area）和平均光密度（density）测量发现，COX-2shRNA治疗组(46142.87±17992.27, 6036.55±3528.01)纤维化较模型组(115630.60±72288.71, 27043.47±16828.40)及空载体组(109433.87±74513.32 32412.22±30425.48)轻。COX-2shRNA抑制肝星状细胞增殖/诱抑制肝星状细胞凋亡的机制可能与神经酰胺途径有关。本研究发现，COX-2shRNA沉默COX-2基因，肝星状细胞神经酰胺合成酶LASS2表达增强，抑制神经酰胺合成酶LASS2，可以明显降低COX-2shRNA 引起的HSC-T6细胞内的神经酰胺水平升高。（2）诱导肝星状细胞衰老。动物及细胞实验证实，COX-2shRNA沉默COX-2基因具有促进纤维化肝组织肝星状细胞衰老的作用；细胞实验证实，COX-2shRNA诱导HSC-T6衰老相关基因p53、p21基因表达增加；Etoposide诱导肝星状细胞衰老可抑制肝星状细胞增殖；COX-2shRNA诱导诱导肝星状细胞衰老可能与p53 信号途径有关。本研究对于揭示COX-2参与肝纤维发生发展机制，及为肝纤维化防治提供新的思路具有一定的意义。