Notch1通过非经典的Notch信号通路调节胰腺星形细胞的活化

Notch1 regulates activation of pancreatic stellate cells by non-classical Notch signaling pathway

目的:探讨Notch1在胰腺星形细胞(pancreatic stellate cells,PSCs)活化中的作用。方法:利用免疫组织化学法与免疫荧光双标法检测Notch1在人胰腺导管腺癌(pancreatic ductal adenocarcinoma,PDAC)组织的表达情况;原代分离培养小鼠PSCs,利用油红O染色、Western blot及RT-qPCR法对其进行鉴定,并利用Western blot及RT-qPCR检测Notch1及其下游关键分子HES1的表达情况;转染Notch1小干扰RNA(Notch1 siRNA)至小鼠PSCs后,利用Western blot检测α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、纤连蛋白(fibronectin)、I型胶原(collagen typeⅠ,ColⅠ)、Notch1及HES1的表达情况;利用划痕实验与CCK-8实验检测Notch1 siRNA对小鼠PSCs迁移与细胞活力的影响。结果:免疫组化与免疫荧光双标染色结果显示,Notch1表达在α-SMA阳性的PDAC间质细胞中;成功培养了小鼠PSCs细胞,且活化的PSCs中α-SMA、fibronectin和ColⅠ的表达升高,Notch1与HES1的表达均高于未活化的PSCs(P<0.01);转染Notch1 siRNA至小鼠PSCs后,细胞中α-SMA和ColⅠ的表达显著降低,而fibronectin和HES1的表达无显著改变;敲减活化的PSCs中Notch1的表达后,细胞的活力及迁移能力显著降低。结论:Notch1参与调节PSCs的活化,抑制Notch1的表达可抑制活化PSCs标志物α-SMA和ColⅠ的表达,降低PSCs的活化程度、活力及迁移能力,并且Notch1调节PSCs的活化不依赖经典的Notch信号通路。

AIM:To investigate the effect of Notch1 on the activation of pancreatic stellate cells(PSCs).METHODS:The expression of Notch1 in pancreatic duct adenocarcinoma(PDAC)tissues was detected by the immunohistochemical and immunofluorescence double staining.The PSCs were isolated,cultured,and identified by oil red O staining,Western blot and RT-qPCR.The expression of Notch1 and HES1 was detected by Western blot and RT-qPCR.After transfection of Notch1 siRNA to PSCs,Western blot was used to detect the protein expression ofα-smooth muscle actin(α-SMA),fibronectin and collagen typeⅠ(ColⅠ)in activated PSCs.The expression of Notch1 and HES1 was also detected by Western blot.The effects of Notch1 siRNA on migration ability and viability of PSCs were determine by scratch test and CCK-8 assay.RESULTS:The results of immunohistochemical and immunofluorescence double staining showed that Notch1 expressed inα-SMA positive cells in PDAC stroma.The mouse PSCs were successfully cultured,and the expression ofα-SMA,fibronectin,ColⅠ,Notch1 and HES1 in activated PSCs were significantly increased compared with unactivated PSCs(P<0.01).After transfection of Notch1 siRNA to mouse PSCs,the expression ofα-SMA and ColⅠwas significantly reduced compared with negative groups,but the expression of fibronectin and HES1 did not change significantly.After knock-down of Notch1 expression in activated PSCs,the migration ability and viability of PSCs were significantly reduced compared with negative group.CONCLUSION:Notch1 is involved in regulating the activation of PSCs.Knock-down of Notch1 expression inhibits the expression of the markers of activated PSCs,α-SMA and ColⅠ,reduces the activation of PSCs,and attenuates the migration capacity and viability of PSCs.Notch1 regulates the activation of PSCs without relying on the classic Notch signaling pathway.