摘要
目的通过siRNA沉默小鼠胰腺星状细胞中STAT3基因,探讨STAT3信号转导通路在慢性胰腺炎胰腺纤维化中的作用。方法根据Gene Bank中STAT3全长c DNA序列,选取特异性序列为干扰作用的靶点,构建STAT3siRNA表达载体,脂质体介导转染PSC,ELISA法检测PSC产生的趋化因子,MTT法检测胰腺星状细胞增殖,细胞爬片免疫组化染色检测α-SMA阳性细胞,Western blot和RT-PCR检测胰腺星状细胞STAT3蛋白及mRNA表达水平,在确定胰腺星状细胞内STAT3基因成功沉默的基础上,给予TGF-β1刺激PSC。结果和对照组相比,ELISA法,MTT法和细胞爬片免疫组化显示各时间点,诱导组、空载组和转染组A值,α-SMA,IL-6和MCP-1表达量均有所升高(P<0.01),转染组A值,α-SMA,IL-6和MCP-1表达量明显低于诱导组和空载组(P<0.01),诱导组和空载组A值,α-SMA、IL-6和MCP-1表达比较差异无统计学意义(P>0.05)。PCR和Western blot结果显示,和对照组相比,诱导组、空载组和转染组α-SMA和Smad2表达明显升高(P<0.01),p-Smad2和PPAR-γ的表达明显降低(P<0.01),转染组α-SMA和Smad2的表达明显低于导组和空载组(P<0.01),p-Smad2和PPAR-γ表达明显高于诱导组和空载组(P<0.01)。转染组STAT3和p-STAT3表达明显低于对照组、诱导组和空载组(P<0.01)。结论siRNA通过调控STAT3信号通路介导下游靶基因的表达,参与抑制胰腺星状细胞活化、进而对抗胰腺纤维化。这一结果为慢性胰腺炎的治疗提供了新的治疗靶点。
Objective To explore the role of STAT3 signal transduction pathway in pancreatic fibrosis in chronic pancreatitis by silencing STAT3 gene in mouse pancreatic stellate cells by siRNA.Methods According to the full-length cDNA sequence of STAT3 in GeneBank,the specific sequence was selected as the target of interference,the STAT3 siRNA expression vector was constructed,the PSC was transfected by liposome,the chemokine produced by PSC was detected by ELISA,and the pancreatic stellate cells was detected by MTT assay.Stellate cell proliferation,cell culture slide immunohistochemical staining forα-SMA positive cells,Western blot and RT-PCR detection of pancreatic stellate cells STAT3 protein and mRNA expression levels,based on the successful silencing of STAT3 gene in pancreas,TGF-β1 was given to stimulate PSC.Results ELISA,MTT assay and cell slide immunohistochemistry showed that the A values ofα-SMA,IL-6 and MCP-1 were higher in the induction group,the empty group and the transfection group at each time point.(P<0.01),the expression of A,α-SMA,IL-6 and MCP-1 in the transfection group were significantly lower than those in the induction group and the empty group(P<0.01),and the A value of the induction group and the empty group.There were no significant difference in the expressions of SMA,IL-6 and MCP-1(P>0.05).The expressions ofα-SMA and Smad2 in the control group were significantly lower than those in the control group,the empty group and the transfection group(P<0.01).The expressions of PPAR-γwere significantly higher than that of the induction group,the empty group and the transfection group.The expressions ofα-SMA and Smad2 in the transfection group were significantly lower than those in the control group and the empty group(P<0.01),and the expression of PPAR-γwas significantly higher than that in the induction group and the empty group(P<0.01).The expressions of STAT3 and p-STAT3 in the transfected group were significantly lower than those in the control group,the induction group and the empty group(P<0.
作者
陈瑜
吴世卫
马晓军
CHEN Yu;WU Shi-wei;MA Xiao-jun(College of Basic Medicine,Shanxi University of Traditional Chinese Medicine,Xianyartg,Shanxi 712046,China)
出处
《毒理学杂志》
CAS
CSCD
2019年第6期454-460,453,共7页
Journal of Toxicology
基金
陕西省教育厅科学研究计划项目(11JK0717)
