CCl4诱发galectin-3基因敲除鼠急性肝损伤中GRP78和BAD表达

目的观察半乳糖凝集素-3(galectin-3)基因敲除鼠肝脏中GRP78和BAD表达,探讨急性肝损伤中galectin-3抗凋亡作用。方法制备GRP78和BADDNA探针,分别利用Western和Northern杂交技术检测在CCl4损伤galectin-3基因敲除鼠肝组织中GRP78和BAD蛋白和mRNA表达。结果成功构建PTS1-BAD和PTS1-GRP78扩增质粒。GRP78蛋白主要定位在微粒体中,galectin-3 / 小鼠经CCl4处理6-14h后,肝脏GRP78表达上调至高峰,而后恢复正常;在galectin-3-/-小鼠中,CCl4处理后GRP78表达水平未见变化。正常或者CCl4处理的galectin-3 / 和-/-小鼠中GRP78 mRNA表达未见显著差别。galectin-3 / 鼠中主要定位在微粒体中在CCl4处理前后未见BAD蛋白在细胞浆和微粒体中表达。galectin-3-/-中BAD主要定位在肝细胞浆和微粒体,CCl4处理前后表达升高。BAD mRNA 1.6kb片段在galectin-3 / 和-/-小鼠经CCl4处理前后均没有变化,但0.9kb片段galectin-3 / 和-/-小鼠经CCl4处理后表达降低。结论CCl4可诱导肝脏mRNA降解,该过程中参与细胞凋亡的蛋白质表达升高。galectin-3 / 小鼠CCl4处理后肝细胞凋亡以内质网应急途径为主,而galectin-3-/-则以线粒体为主。在肝细胞中galectin-3对线粒体凋亡和内质网应急途径均有抑制作用。

Expression of GRP/8 and BAD in galectin-3 Knock-out Mouse Liver Injured by CCl4

Objective To observe GRP78 and BAD expression in galectin-3 / and-/-mouse livers injured by CCl_4 and explore the anti-apoptotic role of galectin-3 in hepatocyte injury.Methods The DNA probes of GRP78 and BAD were prepared.The expression of GRP78 and BAD in galectin-3 / and-/-mouse liver injured by CCl_4 at the levels of protein and mRNA was determined by Western or Northern blot respectively.Results PTS1-BAD and PTS1-GRP78 plasmid DNAs were successfully constructed and amplified.GRP78 protein was mainly localized in microsome of hepatocytes,and its expression was increased to the peak after 6-14 h of CCl_4 administration,and then decreased to normal in galectin-3 / mouse liver.No change in GRP78 expression level was observed after CCl_4 administration in galectin-3-/-mouse liver.GRP 78 mRNA expression was not altered at 24 h after CCl_4 administration in galectin-3 / and-/-mice.BAD protein was localized in cytosol and microsome of hepatocytes and no expression was found in galectin-3 / mouse liver even after CCl_4 treatment.It was highly expressed in galectin-3-/-mouse liver after CCl_4 administration.BAD expression was higher in galectin-3-/-mice than that in glaectin-3 / ones.1.6 kb BAD mRNA expression didn't show alteration in galectin-3 / and-/-mouse liver with or without CCl_4 administration,while 0.9kb BAD mRNA expression was decreased after CCl_4 treatment.Conclusion CCl_4 could degrade liver mRNA and induce synthesis of apoptosis-related protein.The ER-stress-induced apoptosis mainly occurs in galectin-3 mouse liver after CCl_4 treatment,whereas mitochondial apoptosis in galectin-3-/-one.Galectin-3 can inhibit mitochondrial apoptosis and apoptosis induced by ER-stress.