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目的研究姜黄素对肝星状细胞(HSC)增殖活化的影响与过氧化物酶体增殖因子活化受体γ(PPARγ)之间的关系。方法2005年4月至2006年8月,上海中医药大学曙光医院肝病研究所采用肝脏原位灌流酶消化、Nycodenz密度梯度离心法分离培养大鼠HSC,使用药物处理细胞,MTT法检测药物对细胞增殖的影响。收集裂解细胞抽提细胞总RNA,半定量RT-PCR检测基因表达水平。抽提细胞总蛋白,10%聚丙烯酰胺凝胶电泳分离蛋白,Westernblot检测蛋白表达水平。结果姜黄素在10~50μmol/L范围内呈剂量依赖性抑制HSC的增殖。姜黄素对HSC增殖的抑制作用可以被PPARγ特异性拮抗剂GW9662所阻断。在原代培养第1、4、7天和传代培养的HSC中,PPARγ基因表达水平随着HSC活化程度的增加而不断下降;姜黄素显著上调PPARγ表达水平,这种作用能够被GW9662阻断。姜黄素能够在基因和翻译水平显著抑制α平滑肌肌动蛋白的表达,GW9662能够阻断姜黄素的抑制作用。结论姜黄素通过上调PPARγ抑制大鼠HSC的增殖与活化。
Objective To study the effect of curcumin on the proliferation and activation of hepatic stellate cells (HSC) and the relationship between peroxisome proliferator-activated receptor γ (PPARγ). Methods From April 2005 to August 2006, the Institute of Liver Diseases, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine used in situ liver perfusion digestion and Nycodenz density gradient centrifugation to isolate and culture rat HSCs. Cells were treated with drugs and MTT assay was used to detect drug-to-cells. The effect of proliferation. The lysed cells were collected for total RNA extraction and semi-quantitative RT-PCR was used to detect gene expression levels. Total cell protein was extracted and protein was separated by 10% polyacrylamide gel electrophoresis. Protein expression level was detected by Western blot. Results Curcumin inhibited the proliferation of HSC in a dose-dependent manner in the range of 10 to 50 μmol/L. The inhibitory effect of curcumin on HSC proliferation can be blocked by PPARγ-specific antagonist GW9662. In the first, fourth and seventh days of primary culture and HSC passaged, the expression level of PPARγ decreased with the increase of the activation of HSC. Curcumin significantly up-regulated the expression of PPARγ, and this effect could be blocked by GW9662. Curcumin can significantly inhibit the expression of α smooth muscle actin at the level of gene and translation, and GW9662 can block the inhibitory effect of curcumin. Conclusion Curcumin can inhibit the proliferation and activation of rat HSC by up-regulating PPARγ.