关键词: 受体, 鞘磷脂, RNA, 小分子干扰, 涎腺, 腺泡细胞, 遗传载体

Abstract:

Objective：To construct sphingosine 1-phosphate receptor-1 (S1P1)-small interfering RNA (siRNA) lentiviral vectors and infect human salivary gland cells (HSG), and to investigate its possible therapy on Sj-gren’s syndrome. Methods: HSG cells were divided into blank group, empty vector group, scramble-siRNA group and S1P1-siRNA group. The lentiviral vectors expressing siRNA against S1P1 and the pLL3.7 were respectively transfected into 293T cells with pMD2.G, pMDL g/p RRE, pRSV-REV to produce virus, and then infect HSG cells. The efficiency was observed by flow cytometry after the transfection for 48 h. The expression levels of S1P1 mRNA of HSG were detected by real-time RT-PCR and the expression of S1P1 protein was detected by immunohistochemistry method. The expression levels of interferon-γ (IFN-γ) and interleukin (IL)-17 in the supernatant of the cells were detected by ELISA method. Results:(1) The scramble-siRNA, S1P1-siRNA lentiviral vector was successfully constructed, and the lentivirus titer was about 3.5×108  TU/mL. (2) The level of S1P1 mRNA was lower in S1P1-siRNA group than those in the blank group, empty vector group, and scramble-siRNA group 48 h after infection, there were significant differences between them (P<0.05). (3) The expression of S1P1 protein was lower in S1P1-siRNA group than those in blank group, empty vector group, and scramble-siRNA group 48 h after transfection, there were significant differences between them (P<0.05). (4) The levels of IL-17 were lower in S1P1-siRNA group than those in blank group, empty vector group, and scramble-siRNA group 48 h after transfection, there were significant differences between them (P<0.05). (5) The levels of IFN-γ in S1P1-siRNA group were lower than those in blank group, empty vector group, and scramblesiRNA group 48 h after transfection, there were significant differences between them (P<0.05). Conclusion: The lentiviral vector targeting S1P1 was successfully constructed. S1P1 siRNA could suppress the levels of S1P1 mRNA and protein, and decrease the expression of IL-17 and IFN-γ. S1P1 siRNA could infect HSG cells stably and inhibit the expression of S1P1 gene specifically and efficiently, and reduce the levels of inflammatory cytokines.

Key words: Receptors, lysosphingolipid, RNA, small interfering, Salivary glands, Acinar cells, Genetic vectors