慢病毒介导的脯氨酰寡肽酶过表达对大鼠肝星状细胞功能的影响

The effects of lentivirus-mediated overexpression of prolyloligopeptidase in rat HSC-T6 cells

目的探讨携带脯氨酰寡肽酶(prolyloligopeptidase,POP)基因的慢病毒转染大鼠肝星状细胞系-T6(hepatic stellate cellsT6,HSC-T6)对其生物学功能的影响,以揭示其在肝脏炎症与纤维化发生、发展中的作用。方法采用大鼠HSC-T6作为研究对象,设计合成携带大鼠POP基因的慢病毒,转染HSC-T6;通过ELISA方法检测各组HSC内Ac-SDKP水平;Real-time-PCR法检测各组HSC内相关基因表达量变化(POP、TGF-β1、α-SMA、MCP-1、Col I);Western blotting方法检测各组HSC中相关蛋白表达变化(POP、TGF-β1、α-SMA、Smad7、p-Smad2/3、PPAR-γ);CCK-8和流式细胞术检测转染POP对HSC增殖和凋亡的影响。结果慢病毒能高效感染HSC-T6并表达有活性的POP蛋白,与正常对照组及空病毒组相比,POP慢病毒感染的细胞内Ac-SDKP明显升高,Smad7、PPAR-γ蛋白表达显著升高,而α-SMA表达显著下降;POP慢病毒感染的细胞内MCP-1及α-SMA mRNA表达显著下调;此外,POP可促进HSC-T6增殖生长,而对HSC-T6凋亡无影响。结论 POP在HSC内可能发挥抗炎、抗纤维化作用,其机制可能通过促进Smad7及PPAR-γ的表达,抑制MCP-1及α-SMA的表达而发挥作用。

Objective To investigate the biological effects of lentivirus-mediated overexpression of prolyloligopeptidase(POP)in rat HSC-T6 cells and to elucidate its role in the development of liver inflammation and fibrosis.Methods A recombinant lenti-virus overexpression rat POP was constructed and transfected with HSC-T6.The intracellular level of Ac-SDKP was determined by ELISA.The mRNA levels of POP,TGF-β1,α-SMA,MCP-1 and Col I in HSC-T6 were detected by Real-time-PCR.Western blotting was used to detect the protein levels of POP,TGF-β1,α-SMA,Smad7,p-Smad2/3 and PPAR-γin HSC-T6.The cell proliferation and cell apoptosis were detected by CCK-8 and flow cytometry,respectively.Results HSC-T6 cells were transfected with POP-lenti-virus efficiently and successfully expressed more POP protein.And compared with the normal control cells and mock-transfected cells,the Ac-SDKP level was significantly elevated in POP-lentivirus transfected cells;the protein levels of Smad7 and PPAR-γwere also up-regulated,but the protein level ofα-SMA was down-regulated,the mRNA levels of MCP-1 andα-SMA were down-regulated.In addition,POP promoted the proliferation of HSC-T6 cells but had no effect on the apoptosis of HSC-T6 cells.Conclusion POP may play an anti-inflammatory and anti-fibrosis role in HSC via enhancing the expressions of Smad7 and PPAR-γ,inhibiting the expressions of MCP-1 andα-SMA.