R-脊椎蛋白1对肝星状细胞活化的作用及其机制

Role of R-Spondin1 in the activation of hepatic stellate cells

目的 探讨R-脊椎蛋白1（R-Spondin1）在小鼠肝纤维化模型及肝星状细胞（HSC）中的动态表达情况及其对HSC活化的影响. 方法 体内实验：健康雄性昆明种小鼠24只,随机分为2组.模型组（16只）：用20％四氯化碳（CCl4）橄榄油5ml/kg皮下注射,2次/周;对照组小鼠（8只）不作任何处理.10周后处死小鼠,留取肝组织,Western blot检测R-Spondin1、α-平滑肌肌动蛋白（c-SMA）、胶原蛋白Ⅰ（collagen Ⅰ）的蛋白水平,Real-time PCR检测R-Spondinl、α-SMA、collagenⅠ的mRNA水平.体外实验：分离小鼠HSC,检测R-Spondin1、α-SMA、核内β-catenin的蛋白表达情况,以及R-Spondin1的mRNA水平、T淋巴细胞转录因子（TCF）活性在HSC自发激活前后的变化,并比较小鼠HSC经R-Spondin1和抑癌蛋白刺激前后的活化差异.两组间均数比较采用t检验;多组资料采用单因素方差分析,进一步的两两比较采用Dunnett检验. 结果 与对照组相比,肝纤维化模型组R-Spondin1蛋白和mRNA表达水平明显增高,差异有统计学意义（蛋白相对表达量为3.16±0.18与0.99±0.16,t=13.31,P＜0.01;mRNA相对水平为4.36±0.26与0.98±0.12,t=21.46,P＜0.01）.在HSC的自发激活过程中,核内β-catenin的蛋白水平和TCF活性显著增强（核内β-catenin蛋白相对表达量为4.47±0.21与0.97±0.14,t=25.25,P＜0.01;TCF相对活性为5.33±0.34与1.03±0.09,t=20.93,P＜0.01）;R-Spondin1的蛋白和mRNA表达水平显著上调（蛋白相对表达量为4.54±0.18与1.04±0.12,t=31.17,P＜0.01;mRNA相对水平为5.13±0.15与1.01±0.16,t=38.06,P＜0.01）.α-SMA、核内β-catenin的蛋白表达及TCF活性在R-Spondin1刺激后显著上调;而在R-Spondin1和抑癌蛋白共同刺激后无显著变化. 结论 R-Spondin1可能通过增强β-catenin在细胞核内的积聚影响HSC的活化.

Objective To investigate the role of R-Spondinl in the activation of hepatic stellate cells （HSCs）.Methods Twenty-four healthy male Kunming mice were randomly divided into the following two groups：fibrosis model group （n =16） and control group （n =8）.Hepatic fibrosis was induced by subcutaneous injections of CC14 （20% in olive oil） at a dose of 5 ml/kg twice per week.After 10 weeks,the animals were sacrificed by CO2 over-exposure and liver tissues were harvested.The protein and mRNA levels of R-Spondin1,αt-SMA,and collagen Ⅰ were examined by Western blot assay and real-time PCR respectively.Additionally,HSCs were isolated from the mice liver tissues to examine the time-series expression changes of R-Spondinl,α-SMA,and nuclear β-catenin.TCF activity was analyzed by luciferase reporter assay.Moreover,HSCs were cocultured with recombinant R-Spondin1 and DKK1 to evaluate dose-response.Results R-Spondinl expression was significantly higher in the fibrosis model group than in the control group （protein level：3.16±0.18 vs.0.99±0.16,t =13.31,P 〈 0.01; mRNA level：4.36±0.26 vs.0.98±0.12,t =21.46,P 〈 0.01）.The culture-activated mouse HSCs showed up-regulated TCF activity （5.33±-0.34 vs.non-activated：1.03±0.09,t =20.93,P 〈 0.01）,nuclear β-catenin expression （4.47±0.21 vs.0.97±0.14,t =25.25,P 〈 0.01）,and R-Spondin1 expression （protein level：4.54±0.18 vs.1.04±0.12,t =31.17,P 〈 0.01; mRNA level：5.13±0.15 vs.1.01±0.16,t =38.06,P 〈 0.01）.Exogenous stimulation of freshly isolated mouse HSCs with recombinant R-Spondin1 induced a dose-dependent increase in both TCF activity and the expression of nuclear β-catenin and αt-SMA.DKK1 down-regulated activities of factors in the WNT signaling pathway and repressed activation of HSCs.Conclusion R-Spondin1 may promote HSC activation by enhancing the canonical WNT signaling pathway.