【摘要】：目的探討TOLL樣受體4(TLR4)在脂多糖(LPS)誘導(dǎo)的大鼠肝內(nèi)膽管組織損傷中的作用及機(jī)制。方法將48只SD大鼠隨機(jī)均分為四組,LPS+siRNA轉(zhuǎn)染組和LPS+陰性病毒組經(jīng)尾靜脈分別注入siRNA-TLR4純化腺病毒、陰性對(duì)照病毒,對(duì)照組和LPS組均經(jīng)尾靜脈注射等量生理鹽水。給藥后24 h,LPS組、LPS+siRNA轉(zhuǎn)染組和LPS+陰性病毒組均經(jīng)膽總管內(nèi)緩慢注入LPS,對(duì)照組注入等量生理鹽水。制模后48、72 h各組分別取6只大鼠,采集肝門部包含膽管的肝組織標(biāo)本。采用HE染色觀察肝門部肝內(nèi)膽管組織炎性細(xì)胞浸潤(rùn)情況及膽管形態(tài)等病理變化。采用免疫組化法檢測(cè)膽管上皮細(xì)胞TLR4、細(xì)胞角蛋白19(CK19)、波形蛋白(Vimentin)蛋白表達(dá),采用RT-PCR法檢測(cè)膽管組織TLR4、CK19、Vimentin mRNA表達(dá)。結(jié)果 LPS組造模48、72 h時(shí)膽管周圍炎性細(xì)胞浸潤(rùn)且膽管間質(zhì)水腫,伴周圍纖維結(jié)締組織輕度增生;LPS+陰性病毒組表現(xiàn)與LPS組相似;LPS+siRNA轉(zhuǎn)染組造模48 h時(shí)膽管周圍僅有少量炎性細(xì)胞,造模72 h時(shí)膽管周圍炎性細(xì)胞稍增多,但較LPS組顯著減輕。與對(duì)照組比較,其他三組造模48、72 h時(shí)膽管上皮細(xì)胞CK19蛋白及mRNA表達(dá)均降低,TLR4、Vimentin蛋白及mRNA表達(dá)均升高,組間比較P均0.01;LPS+siRNA轉(zhuǎn)染組造模48、72 h時(shí)膽管上皮細(xì)胞CK19蛋白及mRNA表達(dá)均高于LPS組、LPS+陰性病毒組,TLR4、Vimentin蛋白及mRNA表達(dá)均低于LPS組、LPS+陰性病毒組,組間比較P均0.01。結(jié)論 LPS誘發(fā)肝內(nèi)膽管組織發(fā)生炎癥反應(yīng)及纖維化損傷過(guò)程中產(chǎn)生大量TLR4,TLR4可導(dǎo)致肝膽管上皮細(xì)胞發(fā)生上皮間質(zhì)轉(zhuǎn)化,從而加重纖維化損傷程度。
[Abstract]:Aim to investigate the role and mechanism of TOLL-like receptor 4 (TLR4) in lipopolysaccharide (LPS)-induced intrahepatic bile duct injury in rats. Methods Forty-eight SD rats were randomly divided into four groups:, LPS siRNA transfection group and LPS negative virus group. SiRNA-TLR4 purified adenovirus was injected into the tail vein, and the negative control virus was injected into the tail vein respectively. The control group and the LPS group were injected with the same amount of normal saline through the tail vein. At 24 h after administration, LPS siRNA-transfected group and LPS-negative virus group were injected with the same amount of normal saline into the common bile duct by slow injection into the common bile duct of LPS, control group. At 48 h and 72 h after model, 6 rats in each group were taken and the liver tissue specimens containing bile duct in the hilum were collected. HE staining was used to observe the infiltration of inflammatory cells in the intrahepatic bile duct and the morphological changes of the bile duct. The expression of cytokeratin 19 (CK19) and vimentin (Vimentin) in bile duct epithelial cells (TLR4,) was detected by immunohistochemical method, and the expression of TLR4,CK19,Vimentin mRNA in bile duct tissue was detected by RT-PCR method. Results at 48 h and 72 h after LPS, inflammatory cells infiltrated around the bile duct and edema of the bile duct stroma, with slight proliferation of the surrounding fibrous connective tissue, and the expression of LPS-negative virus group was similar to that of the LPS group. There were only a few inflammatory cells around the bile duct in the LPS siRNA transfection group at 48 h, and a little more inflammatory cells in the bile duct around the bile duct at 72 h after the model was made, but decreased significantly than that in the LPS group. Compared with the control group, the expression of CK19 protein and mRNA in bile duct epithelial cells decreased and the expression of TLR4,Vimentin protein and mRNA increased in the other three groups (P < 0.01). The expression of CK19 protein and mRNA in bile duct epithelial cells of LPS siRNA transfection group was higher than that of LPS group, LPS negative virus group, TLR4,Vimentin protein and mRNA expression of LPS negative virus group were lower than that of LPS group, LPS negative virus group at 72 h after transfection, and the expression of TLR4,Vimentin protein and mRNA was significantly higher than that of LPS negative virus group (P < 0.01). Conclusion the inflammatory reaction of intrahepatic bile duct tissue induced by LPS and the production of a large number of TLR4,TLR4 in the process of fibrosis injury can lead to epithelial stroma transformation of hepatobiliary epithelial cells, thus aggravating the degree of fibrosis injury.
【作者單位】： 河南省省立醫(yī)院;遵義醫(yī)學(xué)院附屬醫(yī)院;
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目(81260085)
【分類號(hào)】：R575.62

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