Feng Yi-fei, Li Tao, Li Shu, Peng Ji-run, Leng Xi-sheng
Department of Hepatobiliary Surgery, Peking University People's Hospital, Beijing 100044, China.
Zhonghua Gan Zang Bing Za Zhi. 2009 Sep;17(9):649-52.
To observe the effects of galectin-3 on proliferation and apoptosis of hepatic stellate cells.
RT-PCR and Western blot were used to detect the expression of galectin-3 in hepatic stellate cells. Short hairpin DNA targeting galectin-3 of rat was was ligated into the recombinant vector pGCsilencer U6/Neo/GFP/shRNA plasmid. Then the plasmid was transfected into rat hepatic stellate cells. RT-PCR and Western blot were used to detect the interfering efficiency. Cell proliferation level was observed by CCK8 method at 24, 48 and 72 hours after transfection. Cell apoptosis was measured by Annexin V/PI-labeled flow cytometric analysis.
Expression of galectin-3 in HSC was verified by both RT-PCR and Western blot. The recombinant vector was successfully constructed and verified, and was transfected into rat hepatic stellate cells. Western Blot and RT-PCR results demonstrated that the expression level of Galectin-3 was significantly down-regulated in galectin-3 shRNA transfected cells compared to control vector transferred cells. CCK8 assay indicated that proliferation of Galectin-3 knockdown cells was lower than that of control cells 48 and 72 hours post-transfection. Apoptotic cells in shRNA-interfering group were higher than those in control group both in early stage and advanced stage.
Hepatic stellate cells can express galectin-3. Inhibition of galectin-3 using RNAi technique can suppress proliferation and induce apoptosis in HSC.
观察半乳糖凝集素-3对肝星状细胞增殖和凋亡的影响。
采用RT-PCR和蛋白质免疫印迹法检测肝星状细胞中半乳糖凝集素-3的表达。将靶向大鼠半乳糖凝集素-3的短发夹DNA连接到重组载体pGCsilencer U6/Neo/GFP/shRNA质粒中。然后将该质粒转染到大鼠肝星状细胞中。采用RT-PCR和蛋白质免疫印迹法检测干扰效率。转染后24、48和72小时，通过CCK8法观察细胞增殖水平。采用膜联蛋白V/PI标记的流式细胞术分析检测细胞凋亡。
RT-PCR和蛋白质免疫印迹法均验证了半乳糖凝集素-3在肝星状细胞中的表达。成功构建并验证了重组载体，并将其转染到大鼠肝星状细胞中。蛋白质免疫印迹和RT-PCR结果表明，与转染对照载体的细胞相比，转染半乳糖凝集素-3短发夹RNA的细胞中半乳糖凝集素-3的表达水平显著下调。CCK8检测表明，转染后48和72小时，半乳糖凝集素-3敲低细胞的增殖低于对照细胞。在早期和晚期，短发夹RNA干扰组的凋亡细胞均高于对照组。
肝星状细胞可表达半乳糖凝集素-3。利用RNAi技术抑制半乳糖凝集素-3可抑制肝星状细胞增殖并诱导其凋亡。
