神经鞘磷脂合成酶基因沉默对细胞凋亡的影响

Influence of Sphingomyelin Synthase Knockdown on Cell Apoptosis

以HEK293细胞为模型,利用RNA干扰技术,将神经鞘磷脂合成酶(SMS)的同工酶(SMS1和SMS2)的siRNA分别联合、转染HEK293细胞.通过薄层层析法评价SMS酶活性,同时测神经酰胺(Cer)、卵磷脂(PC)及神经鞘磷脂(SM)的水平,并以流式细胞仪和AnnexinV-FITC、PI双染法检测细胞凋亡.结果显示,与对照组相比,SMS基因沉默后SMS表达水平降低(分别降低17%,20%,49%);SM水平显著性降低(P<0.05);Cer水平显著性升高(P<0.05);TNF-α诱导的凋亡显著性升高[分别为58%(P<0.01),24%(P<0.05),77%(P<0.01)].这些结果提示,SMS基因沉默能降低SM水平并升高Cer水平,明显增加TNF-α诱导的HEK293细胞凋亡.由于SM是动脉粥样硬化形成的独立危险因子,因此本研究有可能为动脉粥样硬化的治疗找到新的靶点和有效途径.

HEK293 cells were uesd to investigate the relationship between sphingomyelin synthase （SMS） activity and cell apoptosis. SMSlsiRNA and SMS2siRNA were transfected HEK293 cells, respectively, or simultaneously. SMS activity was monitored by thin layer chromatographic assay. Sphingomyelin, ceramide, and phosphatidylcholine levels were determined by an established method. Cell apoptosis was measured by flow cytomertry. The results show that SMS1 and SMS2 knockdown reduces SMS activity（ 17% , 20%, 49%, respectively）, sphingomyelin levels were significantly reduced （P 〈0.05 ）, ceramide was significantly increased （P 〈 0. 05 ）, compared with control group. We then performed cell apoptosis, and found that SMS1, SMS2, and SMS1/SMS2 knockdown significantly induced by TNF-α mediated apoptosis [ 58% （ P 〈 0. 01 ）, 24% （P 〈 0. 05 ）, 77 % （P 〈 0. 01 ）, respectively ], suggesting monitoring SMS activity plays an important role on cell apoptosis, thus may have impact on the development of atherosclerosis.