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目的研究LAIR-1基因沉默对幽门螺杆菌(Helicobacter pylori)诱导单核细胞THP-1凋亡的影响。方法以Hp ATCC26695分别感染THP-1细胞6、12、24和48h,采用流式细胞术检测细胞凋亡及LAIR-1蛋白的表达。应用Lipofectamine RNAiMAX转染3对LAIR-1siRNAs入单核细胞THP-1中,采用半定量RT-PCR及流式细胞术检测沉默效应,筛选出的较有效的一对siRNA再转染入单核细胞THP-1,48h后加入Hp培养24h,采用流式细胞术检测细胞凋亡率。结果 Hp感染THP-1细胞6、12、24和48h后的细胞凋亡率分别为(84±1.77)%、(69.13±4.29)%、(48.3±3.37)%和(21.1±4.67)%,LAIR-1蛋白表达相对荧光强度分别为14 934±178、14 369±244、12 259±523和7438±539;转染后48h较有效的siRNA对LAIR-1mRNA及蛋白沉默效率分别为(39.1±4.67)%和(38.4±3.18)%;将筛选出的siRNA转染细胞48h后,用Hp感染24h,流式细胞仪检测显示转染组与阴性对照组细胞凋亡率分别为(56.1±9.8)%和(36.9±3.2)%,转染后感染组与空白组LAIR-1蛋白表达相对荧光强度分别为12 181±219和10 228±136。结论在Hp感染细胞THP-1 48h内,随着感染时间延长LAIR-1的表达及THP-1细胞凋亡率显著降低即LAIR-1siRNA能抑制LAIR-1的表达及THP-1细胞的凋亡。
Objective To investigate the effect of LAIR-1 gene silencing on the apoptosis of THP-1 cells induced by Helicobacter pylori. Methods THP-1 cells were infected with Hp ATCC26695 for 6, 12, 24 and 48 h, respectively. The apoptosis and the expression of LAIR-1 protein were detected by flow cytometry. Three pairs of LAIR-1siRNAs were transfected into monocytes THP-1 by Lipofectamine RNAiMAX. The silencing effect was detected by semi-quantitative RT-PCR and flow cytometry. The more effective pairs of siRNAs were selected and transfected into monocytes THP-1, 48 h after adding Hp cultured 24h, the apoptosis rate was detected by flow cytometry. Results The apoptosis rates of Hp-infected THP-1 cells were (84 ± 1.77)%, (69.13 ± 4.29)%, (48.3 ± 3.37)% and (21.1 ± 4.67)% at 6, 12, The relative fluorescence intensity of LAIR-1 protein expression was 14 934 ± 178, 14 369 ± 244, 12 259 ± 523 and 7438 ± 539, respectively. The efficiency of LAIR-1 mRNA and protein silencing 48 h after transfection was (39.1 ± 4.67)% and (38.4 ± 3.18)%, respectively. The transfected cells were transfected for 48 hours and then infected with Hp for 24 hours. Flow cytometry showed that the apoptotic rates of transfected cells and negative control cells were (56.1 ± 9.8) ) And (36.9 ± 3.2)%, respectively. The relative fluorescence intensity of LAIR-1 protein expression in infected and blank control groups was 12 181 ± 219 and 10 228 ± 136, respectively. Conclusions The expression of LAIR-1 and the apoptosis rate of THP-1 cells were significantly decreased with the prolongation of infection time within 48 h after THP-1 infection, ie LAIR-1 siRNA could inhibit the expression of LAIR-1 and the apoptosis of THP-1 cells .