活化的肝星状细胞RBP-J可诱导性基因敲除小鼠的构建及应用

Generation and Application of Activated Hepatic Stellate Cell RBP-J inducible Knockout Mice

目的:利用Cre-loxp可诱导系统构建活化的肝星状细胞RBP-J可诱导性基因敲除小鼠模型,以实现肝纤维化过程中活化的肝星状细胞Notch信号通路的特异性阻断。方法:将Sm22α^CreERT2和RBP-J^flox/flox转基因小鼠杂交,得到F1小鼠,将繁殖的F1小鼠继续进行交配,得到F2小鼠,通过PCR鉴定出基因型为Sm22α^Cre ERT2,RBP-J^flox/flox的小鼠。通过腹腔注射四氯化碳建立肝纤维化模型,检测四氯化碳注射不同时间段后肝脏纤维化进展情况和肝星状细胞活化过程中Sm22α的表达情况。注射他莫昔芬诱导活化肝星状细胞中RBP-J基因的特异性敲除。分选肝星状细胞,q-PCR和Western Blot检测活化肝星状细胞中Notch下游靶基因Hes1、Hey1表达情况以验证敲除效果。结果:通过连续交配我们获得了Sm22α^Cre ERT2,RBP-J^flox/flox小鼠。四氯化碳腹腔注射4周即可诱导肝脏发生较为明显的纤维化,同时肝星状细胞活化并逐渐高表达Sm22α。给予他莫昔芬诱导Cre重组酶发挥作用后,敲除了活化的肝星状细胞中的RBP-J基因,其下游基因Hes1、Hey1表达降低70%以上,阻断了Notch信号通路。结论:我们成功构建了RBP-J可诱导性条件性基因敲除小鼠模型,实现了小鼠肝纤维化过程中活化肝星状细胞Notch信号通路的阻断,为深入研究Notch信号通路在肝纤维化中的作用和机制奠定了坚实的基础。

Objective: To generate an inducible mouse line of conditional knockout of RBP-J in activated hepatic stellate cells,thus disrupting the Notch signaling of activated hepatic stellate cells in liver fibrosis. Methods: The Sm22α^Cre ERT2 and RBP-J^flox/flox transgenic mice were crossed. The genotypes of their offspring were determined by polymerase chain reaction analysis and Sm22α^Cre ERT2,RBP-J^flox/floxmice were obtained. Liver fibrosis model was established by intraperitoneal injection of CCl4. Sm22α, a marker of activated hepatic stellate cells during the process of liver fibrogenesis, was evaluated by immunofluorescence. Specific knockout of RBP-J in activated hepatic stellate cells was then realized by intraperitoneal injection of tamoxifen in mice. After purifying hepatic stellate cells from the fibrotic liver, the Notch target genes of Hes1 and Hey1 were examined by using q-PCR and Western-blot. Results: Mice with genotype of Sm22α^Cre ERT2 RBP-J^flox/floxwere obtained by consecutive breeding. Intraperitoneal injection of CCl4 for 4 weeks could establish obvious liver fibrosis. During liver fibrosis, hepatic stellate cells were activated and began to express Sm22α. After intraperitoneal tamoxifen injection, RBP-J was successfully suppressed in Sm22α+ activated hepatic stellate cells as evidenced by significantly reduced expression(more than 70 %) of Notch target genes-Hes1 and Hey1. Conclusions: We have successfully generated an inducible RBP-J conditional knockout mouse line and further developed a model of specific disruption of Notch signaling in activated-HSCs during the process of hepatic fibrogenesis, paving the way for study of the role and mechanisms of Notch signaling in modulating liver fibrosis in the future.