Trps1调控胆管上皮细胞上皮-间质转化的实验研究

Trps1 regulates epithelial-mesenchymal transition of intrahepatic biliary epithelial cells

目的探讨毛发鼻指(趾)综合征1型相关基因(tricho-rhino-pharyngeal syndrome type 1,Trps1)在冷保存再灌注损伤(cold preservation reperfusion injury,CPRI)诱导人肝内胆管上皮细胞(human intrahepatic biliary epithelial cells,HIBECs)上皮-间质转化(epithelial-mesenchymal transition,EMT)中的作用。方法体外模拟CPRI处理HIBECs检测上皮标志物E-cadherin、CK19,间质标志物Vimentin、α-SMA及目标基因Trps1的表达。用Trps1腺病毒或Trps1 siRNA载体转染HIBECs后,重复CPRI诱导实验,探讨Trps1对HIBECs EMT的调控作用。结果随着冷保存时间的延长,上皮标志物E-cadherin、CK19 mRNA和蛋白表达持续减少(P=0.018,0.011;P=0.039,0.044),间质标志物Vimentin、α-SMA表达持续增加(P=0.002,0.026;P=0.035,0.016);冷保存12 h,细胞内Trps1 mRNA和蛋白表达增加(P=0.018,0.031),其后随冷保存时间延长持续减少(P=0.026,0.009)。相关分析显示在该过程中,Trps1与上皮标志物表达正相关(r=0.981,0.913;P=0.000,0.002),而与间质标志物表达负相关(r=-0.711,-0.847;P=0.009,0.005)。Trps1腺病毒转染HIBEC后,重复上述CPRI诱导实验,E-cadherin表达增加(P=0.002,0.000),Vimentin表达减少(P=0.000,0.017),CPRI诱导HIBECs EMT效应受到抑制;Trps1 siRNA转染HIBECs后,重复上述CPRI诱导实验,E-cadherin表达减少(P=0.032,0.044),Vimentin表达增加(P=0.047,0.031),加强CPRI诱导HIBECs EMT效应。结论体外模拟CPRI可诱导HIBECs发生EMT,Trps1参与调控HIBECs EMT,发挥重要的拮抗作用并可能逆转该过程。

Objective To observe the epithelial-mesenchymal transition （EMT） of human intrahepatic biliary epithelial cells （HIBECs） induced by cold preservation reperfusion injury （CPRI）, and to study the role of tricho-rhino-pharyngeal syndrome type 1 （Trpsl） in the occurrence of HIBECs EMT. Methods HIBECs were incubated with simulated CPR1 in vitro, then expressions of E-cadherin, CK19, Vimentin, α-SMA and the target gene Trpsl in HIBECs were detected by RT-PCR and Western blotting after treatment of HIBECs with CPRI. The effect of Trpsl was increased with recombinant adenovirus vector containing Trpsl gene or blocked with Trpsl siRNA, then the role of Trpsl was studied. Results With the prolongation of cold preservation time, the mRNA and protein expression of EMT markers, E-cadherin and CK19 was decreased （P = 0. 018, 0. 011 ; P = O. 039, 0.044 ）, while that of Vimentin and α-SMA was significantly increased （ P = 0. 002, 0. 026 ; P = 0. 035, 0. 016）. The expression of Trpsl mRNA and protein reached its peak at 12 h （P = 0. 018, 0.031 ） and began to decrease at 24 and 48 h, respectively （P =0. 026, 0. 009）. Trpsl was positively correlated with the epithelial markers （r =0. 981,0. 913; P =0. 000, 0.002） but negatively correlated with the mesenchymal markers （ r = - 0.711, - 0. 847 ; P = 0. 009, 0. 005 ）. Recombinant adenovirus vector containing Trpsl gene could up-regulate the CPRI-induced expression decrease of E-cadherin （P = 0. 002, 0. 000） but down-regulate the expression increase of Vimentin （P = 0. 000, 0.017）. Trpsl siRNA could down-regulate the CPRI-induced expression decrease of E-cadherin （P = 0. 032, 0. 044） but up-regulate the expression increase of Vimentin and α-SMA （P = 0. 047, 0. 031）. Conclusion CPRI can induce the occurrence of HIBECs EMT. Trpsl participates in the regulation of HIBECs EMT as a key antagonistic factor, and may reverse the process.