技术专题

复旦大学上海医学院附属中山医院内科肝病科、消化科，美国西奈山医学院老年病研究部的科学家联手在顶级的肝病论文期刊Hepatology发表文章，解析肝细胞toll样受体4单核甘酸多态性与肝硬化间的功能关系。

领导这一研究的是西奈山医学院内科教授、肝病科主任,肝病研究方面世界级权威专家Scott L.Friedman。第一作者是郭津生，来自复旦大学中山医院消化科、复旦大学上海医学院内科学系。

在最近的研究中，科学家们发现纤维化的肝细胞to11样受体4基因（toll-like receptor 4 gene,TLR4)的单核苷酸多态性发生变化，主要的CC等位基因发生变异。研究者对这一单核苷酸多态性进行功能性的研究，并用肝脏stellate细胞作为研究模型。研究者选取两种肝脏stellate细胞（ hepatic ste11ate ce11，HSC），一种是小鼠的TLR4-/-的HSC，一种是人类的HSC细胞系（LX-2），LX-2的特点是与野生型的HSC细胞相比该细胞系的TLR4D299G或T3991互补DNAs与脂多糖具有低反应性。与脂多糖的反应直接关系着肝细胞的炎症反应性。

研究者最后得出结论，TLR4D299G和T399I的SNP与抵抗肝细胞纤维化的有关，并且与诱导TLR4介导的炎症反应与纤维生成信号，降低干细胞凋亡的信号阙值都有相当大的关联。这些研究结果为TLR4 SNP可能是调节肝脏纤维化的理论提供了实质性的分子证据。

Hepatology DOI 10.1002/hep.22697

Functional linkage of cirrhosis-predictive single nucleotide polymorphisms of toll-like receptor 4 to hepatic stellate cell responses
 
Jinsheng Guo 1 2, Johnny Loke 1, Feng Zheng 3, Feng Hong 1, Steven Yea 1, Massayuki Fugita 1, Mirko Tarocchi 1, Olivia T. Abar 4, Hongjin Huang 4, John J. Sninsky 4, Scott L. Friedman 1 *§

1Division of Liver Diseases, Zhongshan Hospital, Department of Internal Medicine, Shanghai Medical College, Fudan University, Shanghai, China
2Division of Digestive Diseases, Zhongshan Hospital, Department of Internal Medicine, Shanghai Medical College, Fudan University, Shanghai, China
3Department of Geriatrics, Mount Sinai School of Medicine, New York, NY
4Celera, Alameda, CA

In a recent study, a single nucleotide polymorphism (SNP) of the Toll-like receptor 4 (TLR4) gene (c.1196C>T [rs4986791, p.T399I]) emerged as conferring protection from fibrosis progression compared to a major, wild-type (WT) CC allele (p.T399). The present study examined the functional linkage of this SNP, along with another common, highly cosegregated TLR4 SNP (c.896A>G [rs4986790, p.D299G]), to hepatic stellate cell (HSC) responses. Both HSCs from TLR4-/- mice and a human HSC line (LX-2) reconstituted with either TLR4 D299G and/or T399I complementary DNAs were hyporesponsive to lipopolysaccharide (LPS) stimulation compared to those expressing WT TLR4, as assessed by the expression and secretion of LPS-induced inflammatory and chemotactic cytokines (i.e., monocyte chemoattractant protein-1, interleukin-6), down-regulation of bone morphogenic protein and the activin membrane-bound inhibitor expression (an inhibitory transforming growth factor  pseudoreceptor), and activation of a nuclear factor B (NF-B)-responsive luciferase reporter. In addition, spontaneous apoptosis, as well as apoptosis induced by pathway inhibitors of NF-B, extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase were greatly increased in HSCs from either TLR4-/- or myeloid differentiation factor 88-/- (a TLR adaptor protein) mice, as well as in murine HSCs expressing D299G and/or T399I SNPs; increased apoptosis in these lines was accompanied by decreased phospho-ERK and Bcl-2. Conclusion: TLR4 D299G and T399I SNPs that are associated with protection from hepatic fibrosis reduce TLR4-mediated inflammatory and fibrogenic signaling and lower the apoptotic threshold of activated HSCs. These findings provide a mechanistic link that explains how specific TLR4 SNPs may regulate the risk of fibrosis progression. (HEPATOLOGY 2009.)