RhoA因子在单增李斯特菌侵袭HepG2细胞中的作用

Role of RhoA in Listeria monocytogenes invasion

目的研究HepG2细胞的Rho家族RhoA因子在Lm侵袭中的作用。方法构建真核表达载体p3×FLAGMyc-CMV-24-RhoA,经脂质体法转染HepG2细胞,使HepG2细胞过表达RhoA;同时针对RhoA序列合成siRNA,脂质体法转染HepG2细胞,抑制HepG2细胞RhoA表达。2种细胞同时感染Lm,通过Real-time PCR检测HepG2细胞RhoA上调/下调时培养物中Lm-Hly基因拷贝数,评估Lm数量。结果HepG2细胞转染p3×FLAG-Myc-CMV-24-RhoA载体后,Western blotting法检测RhoA表达量升高,且48h升高显著,此时HepG2细胞内Lm数量明显低于对照组;HepG2细胞转染siRNA后,Western blotting法检测RhoA表达量降低,且72h降低显著,此时HepG2细胞内Lm数量明显高于对照组。结论Lm侵入受HepG2细胞RhoA因子调控,主动下调细胞RhoA因子表达可能是Lm侵袭机制之一。

Listeria monocytogenes（Lm）is a ubiquitous gram-positive bacterium that is obligate intracellular pathogen.The invasion strategy is depending on host cell cytoskeleton rearrangement.In this study,we explored the role of RhoA in Lminvasion.Eukaryotic over-expressing vector p3×FLAG-Myc-CMV-24-RhoA was constructed,and transfected into HepG2 cells via lipodosome.The specific siRNA targeting to RhoA sequence was synthesized and transfected into HepG2 cells via lipodosome.After transfection,the two group cells were infected with Lmrespectively,then the copy of Hly which was equal to the number of Lm was determined by real-time PCR assay.Results showed that HepG2 cell RhoA expression had a significant increase in 48 hours,and the number of Lminvaded into HepG2 cells was significantly lower than that in the normal cells,after transfected with over-expressing vector p3×FLAG-Myc-CMV-24-RhoA.After transfected with siRNA trageting to RhoA sequence,HepG2 cell RhoA expression had a significant decrease in 72 hours,and the number of Lminvaded into HepG2 cells was significantly higher than that in the normal cells.It＇s indicated that a successful Lminvasion depended on host cell RhoA regulation,and down-regulating host cell RhoA expression might be a strategy for Lminvasion.