Notch3通过TGFβ1信号通路参与调节小鼠胰腺星状细胞的激活  

Notch3 enhances migration and viability of mouse pancreatic stellate cells via TGFβ1 signaling pathway

作　　者：宋海岩[1] 张毅敏[1] 孙春莉 SONG Hai-yan;ZHAG Yi-min;SUN Chun-li(School of Basic Medical Sciences,Xinxiang Medical University,Xinxiang 453003,China)

机构地区：[1]新乡医学院基础医学院,河南新乡453003

出　　处：《中国病理生理杂志》2022年第6期1083-1090,共8页Chinese Journal of Pathophysiology

基　　金：新乡医学院博士科研启动基金资助项目(No.XXBSKYZZ201821);新乡医学院基础医学院培育项目(No.JCYXYJX201903)。

摘　　要：目的:探讨Notch3在小鼠胰腺星状细胞(pancreatic stellate cells,PSCs)激活中的作用机制。方法:原代分离培养小鼠PSCs并用油红O染色鉴定,细胞分组为空白对照组、阴性对照siRNA组、Notch3 siRNA组、转化生长因子β1(transforming growth factorβ1,TGFβ1)组及Notch3 siRNA+TGFβ1组。应用RNA测序检测Notch3基因对其他信号通路的影响;应用免疫组织化学检测Notch3与TGFβ1在人胰腺导管腺癌(pancreatic ductal adenocarcino⁃ma,PDAC)间质及癌旁组织中的表达情况;应用免疫荧光双标法检测Notch3和TGFβ1在PDAC间质的共表达情况;应用Western blot检测Notch3和TGFβ1在空白对照组和Notch3 siRNA组小鼠PSCs中的表达;应用Western blot检测Notch3基因敲减和TGFβ1诱导对活化的PSCs中α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、纤连蛋白(fibronectin)和I型胶原(collagen type I,Col I)蛋白表达的影响;应用划痕实验和CCK-8实验检测Notch3基因敲减和TGFβ1诱导对小鼠PSCs迁移和活力的影响。结果:RNA测序和Western blot结果显示,敲减小鼠PSCs中Notch3基因表达可抑制TGFB1基因和TGFβ1蛋白表达;免疫组化和免疫荧光双标染色结果显示,Notch3和TGFβ1共表达于人PDAC间质细胞中;与Notch3 siRNA组相比,Notch3 siRNA+TGFβ1组小鼠PSCs中fibronectin和Col I蛋白表达显著升高(P<0.05),且PSCs活力和迁移能力显著提高(P<0.01)。结论:抑制小鼠PSCs中Notch3基因的表达,同时给予TGFβ1诱导,可提高小鼠PSCs的迁移能力和活力。Notch3对PSCs激活的调节作用部分依赖TGFβ1信号通路。AIM:To investigate the role of Notch3 in the migration and viability of mouse pancreatic stellate cells(PSCs).METHODS:Mouse PSCs were isolated and cultured.After identified by oil red O staining,the mouse PSCs were divided into mock group,negative control siRNA group,Notch3 siRNA group,transforming growth factorβ1(TGFβ1)group and Notch3 siRNA+TGFβ1 group.The effects of Notch3 gene on other signaling pathways in mouse PSCs were evaluated by RNA sequencing.The expression of Notch3 and TGFβ1 in human pancreatic ductal adenocarcinoma(PDAC)stroma and para-cancerous tissue was detected by the immunohistochemical staining.Co-expression of Notch3 and TGFβ1 in human PDAC stroma was confirmed by immunofluorescence double staining.Western blot was used to de⁃tect Notch3 and TGFβ1 expression.The expression ofα-smooth muscle actin(α-SMA),fibronectin and collagen type I(Col I)were detected using Western blot.The effects of Notch3 knockdown and TGFβ1 on the migration and viability of mouse PSCs were tested using scratch test and CCK-8 assay.RESULTS:The results of RNA sequencing and Western blot demonstrated that inhibition of Notch3 gene inhibited TGFB1 gene and TGFβ1 protein expression in mouse PSCs.Notch3 and TGFβ1 co-expressed in human PDAC stroma cells.TGFβ1 rescued the expression of fibronectin and Col I,and enhanced the migration and viability of mouse PSCs induced by Notch3 siRNA.CONCLUSION:Notch3 regulates the activation,migration and viability of mouse PSCs partially through the TGFβ1 signaling pathway.

关 键 词：Notch3蛋白 转化生长因子β1 胰腺导管腺癌 胰腺星状细胞 

分 类 号：R735.9[医药卫生—肿瘤] R363.21[医药卫生—临床医学]