RNAi对细胞因子信号转导抑制因子1在内皮细胞中表达的沉默作用

背景：利用RNAi技术引发细胞因子信号转导抑制因子1沉默可促进神经内分泌肿瘤细胞的凋亡，然而对于内皮细胞是否有类似的作用，目前尚不清楚。目的：实验拟验证内皮细胞中是否有细胞因子信号转导抑制因子1表达，并在所设计的siRNA中，筛选出1对对细胞因子信号转导抑制因子1沉默效率最高的siRNA。设计、时间及地点：随机对照，体外细胞学实验，于2007-12/2008-06在南昌大学医学院第二附属医院所属的江西省分子医学重点实验室完成。材料：人脐静脉内皮细胞由南京凯基生物公司提供。方法：设计并化学合成4对靶向细胞因子信号转导抑制因子1的siRNA：siRNA-1、siRNA-2、siRNA-3、siRNA-4。培养人脐静脉内皮细胞，利用RT-PCR检测人脐静脉内皮细胞是否有细胞信号转导抑制因子1表达。将带有荧光标记的阴性对照siRNA配制成25，50，75，100 nmol/L的4组，利用脂质体转染细胞，并在荧光倒置显微镜下观察转染效率，对比得出有最佳转染效率的浓度。然后，实验根据不同的处理因素分成8组：siRNA-1、siRNA-2、siRNA-3、siRNA-4和阳性对照组、阴性对照组、转染试剂组、空白对照组4个对照组。将均为有最佳转染效率浓度的各组siRNA转染细胞，48 h后提取RNA。主要观察指标：利用RT-PCR测定细胞因子信号转导抑制因子1 mRNA的相对表达水平。结果：人脐静脉内皮细胞高表达细胞因子信号转导抑制因子1。siRNA在50 nmol/L时有较高的转染效率。4对siRNA干扰后，细胞因子信号转导抑制因子1 mRNA的表达水平与阴性对照、转染试剂组和空白对照组相比明显下调(P < 0.05)，与阳性对照组相比显著下调(P < 0.01)，其中siRNA-3的沉默效率最高(P < 0.05)，阴性对照、转染试剂组和空白对照组与阳性对照组相比明显下调(P < 0.05)，阴性对照、转染试剂组和空白对照组之间差异无显著性意义(P > 0.05)。结论：内皮细胞中存在细胞因子信号转导抑制因子1表达。RNAi抑制细胞因子信号转导抑制因子1在内皮细胞中的表达，实验筛选出1对沉默效率最高的siRNA。

Silencing effect of RNA interference on suppressor of cytokine signaling-1 in human umbilical vein endothelial cells

BACKGROUND: Suppressor of cytokine signaling-1 (SOCS1) silencing by RNAi can induce apoptosis of neuroendocrine tumor cells. However, it is not clear whether apoptosis of endothelial cells can be enhanced by silencing of SOCS1 similarly.OBJECTIVE: To determine whether SOCS1 is expressed in endothelial cell, in addition, to select the most extremely silences SOCS1 from the siRNAs designed. DESIGN, TIME AND SETTING: The in vitro control cell experiment was performed at the Jiangxi Province Key Laboratory of Molecular Medicine, the Second Affiliated Hospital of Nanchang University Medical College from December 2007 to June 2008.MATERIALS: Human umbilical vein endothelial cell (HUVEC) lines were provided by NanJing KeyGen Biotech Co., Ltd. METHODS: HUVEC was cultured. RT-PCR was used to determine whether SOCS1 was expressed in HUVEC. HUVEC were divided into the positive control (GAPDH-siRNA-PC), the negative control (siRNA-NC), the mock control (Mock) and the blank control groups. Four different siRNAs were designed and synthesized; they were siRNA-1, siRNA-2, siRNA-3, siRNA-4, and then transfected with liposome. The negative control siRNAs labelled with fluorescence were transfected with different concentrations (25, 50, 75, 100 nmol/L). Then fluorescent microscope was employed to determine the concentration at which siRNAs were transfected most successfully. RNAs were extracted from siRNAs groups, which had optimum transfection efficiency after 48 hours.MAIN OUTCOME MEASURES: RT-PCR was employed to assess the expression of SOCS1 at gene level.RESULTS: SOCS1 were highly expressed in HUVEC. It was in the 50 nmol/L group that the siRNAs were transfected most successfully. The expressions of SOCS1 in the siRNA-1, siRNA-2, siRNA-3 and siRNA-4 group were reduced compared to the siRNA-NC, Mock and the blank group (P < 0.05). Meanwhile, they were significantly down-regulated compared to the GAPDH-siRNA-PC (P < 0.01). In addition, siRNA-3 had the best silencing efficiency in the four different siRNAs designed group (P < 0.05). The expressions of SOCS1 in the siRNA-NC, Mock and blank group were reduced compared to the GAPDH-siRNA-PC group (P < 0.05). There was no statistical significance among the siRNA-NC, Mock and the blank group (P > 0.05). CONCLUSIONS: SOCS1 is expressed in endothelial cells .The expression of SOCS1 in the endothelial cell can be inhibited by RNA interference. One siRNA with a maximal inhibition rate was selected.