1型1-磷酸鞘氨醇受体的siRNA对人涎腺导管上皮细胞的作用

Effect of type 1 sphingosine-1-phosphate receptor siRNA on human salivary gland cells

目的:构建靶向1型1-磷酸鞘氨醇受体(sphingosine 1-phosphate receptor-1,S1P1)基因的小干扰RNA(small interfering RNA,siRNA)慢病毒表达载体,感染干燥综合征细胞模型——人涎腺导管上皮细胞(human salivary gland cells,HSG),探讨S1P1的siRNA治疗干燥综合征的可能性,为临床靶标治疗提供广阔的思路。方法:实验分空白组、空载体组、scramble-siRNA组及S1P1-siRNA组,分别将p LL3.7空载体、构建成功的scramble-siRNA及S1P1-siRNA慢病毒表达载体与p MD2.G、p MDL g/p RRE、pRSV-REV共转染293T细胞制备病毒,感染HSG细胞株48 h,流式细胞术检测其感染效率,实时荧光定量RT-PCR法检测HSG细胞中S1P1 mRNA的表达水平,细胞免疫组织化学法检测细胞中S1P1蛋白的表达水平,ELISA法检测细胞上清中γ-干扰素(interferon-γ,IFN-γ)和白细胞介素(interleukin,IL)-17的表达水平。结果:(1)成功构建scramble-siRNA、S1P1-siRNA慢病毒表达载体,慢病毒的滴度约为3.5×108TU/m L。(2)感染48 h后S1P1-siRNA组HSG细胞中S1P1 mRNA的表达水平明显低于空白组、空载体组和scramble-siRNA组,差异有统计学意义(P<0.05)。(3)S1P1-siRNA组HSG细胞中S1P1蛋白的表达水平低于空白组、空载体组和scramble-siRNA组,差异有统计学意义(P<0.05)。(4)S1P1-siRNA组HSG细胞分泌的IL-17的浓度明显降低,与空白组、空载体组和scramble-siRNA组比较差异有统计学意义(P<0.05)。(5)S1P1-siRNA组细胞上清液中IFN-γ的浓度降低,与空白组、空载体组和scramble-siRNA组比较差异有统计学意义(P<0.05)。结论:成功构建了靶向S1P1基因的慢病毒载体,S1P1 siRNA可以使S1P1 mRNA和蛋白表达水平下降,细胞上清液中IL-17和IFN-γ的浓度下降,证明S1P1 siRNA可转染HSG细胞且特异、高效地抑制S1P1基因的表达,抑制细胞上清液中细胞因子的表达,为治疗干燥综合征奠定了实验基础。

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 p LL3. 7 were respectively transfected into 293 T cells with p MD2. G,p MDL 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 × 10^8 TU / m L.（ 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 expres-sion 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.