中文摘要：

目的：探讨酸性神经磷脂酶（acid sphingomyelinase，ASMase）在维生素E琥珀酸酯（vitamin E succinate，VES）诱导胃癌细胞凋亡过程中对内质网应激的影响。方法：常规体外培养人低分化胃癌SGC-7901细胞，将细胞分为ASMase抑制剂地昔帕明（desipramine，DES）处理组（12．5μmol／LDES作用2h），对照组（含2％胎牛血清的RPMI-1640培养液），VES＋DES组（用12．5μ1130I／LDES抑制ASMase活性2hA加20μg／mLVES）以及VES处理组（20μg／mL）。以ASMase活性试剂盒检测0、1．5、3、6h时VES＋DES组及VES处理组细胞中ASMase活性。MTT法分别检测12、24和48h时各组细胞的增殖情况。再在处理细胞24h后，采用倒置显微镜观察各组细胞形态；流式细胞仪检测细胞凋亡率；Westernblot法检测细胞中内质网应激相关蛋白GRP78、GRP94、Perk、p—Perk、Chop和Caspase一4的表达。结果：VES（20μg／mE）处理能够诱导细胞中ASMase活性增加，在3h时细胞中ASMase活性达到最高，随后逐渐降低；VES＋DES组细胞中ASMase活性始终维持在较低的水平。与对照组相比，DES组在各项检测中均无明显变化，而VES组与VES＋DES组在处理细胞12、24与48h后，细胞的增殖受到明显抑制（P均〈0．05），并呈时间一效应趋势，且VES＋DES组细胞不同时间点的相对增殖率均明显高于VES组均〈O．05）；细胞形态学显示20μg／mLVES处理细胞后，镜下死细胞明显增多，而VES＋DES组死亡细胞数量较VES组减少；流式细胞术检测凋亡率显示VES处理组为39．21％±1．90％，明显高于空白对照组（3．91％±1．68％）与DES组（4．07％4-1．39％）（P均〈0．05），VES＋DES组（19．47％-4-4．46％）较VES组明显降低（P〈0．05）；Westernblot结果显示VES＋DES组细胞中内质网应激相关蛋白GRP78、GRP94、p-Perk、Chop以及c—Caspase-4表达水平较单纯VES处理组降低fP均〈0．05）。结论：ASMase参与VES通?

英文摘要：

OBJECTIVE：To study the effect of acid sphingomyelinase （ASMase） on endoplasmic reticulum stress （ERS） in vitamin E succinate（VES）-induced apoptosis in human gastric cancer ceils. METHODS： SGC-7901 human gastric cancer ceils were cultured in vitro. The cells were divided into four groups： desipramine （DES） group （cells were treated at 12.5μmol/L for 2 h）, control group （containing 2% FBS RPMI-1640 culture medium）, VES＋ DES group （ceils were treated with DES at 12.5 ~ mol/L for 2 h, then with VES at 20 I~ g/ml） and VES group （20 p, g/ml）. ASMase activity detection kit was used to assess the ASMase activity of VES and VES ＋ DES groups at 0, 1.5, 3 and 6 h. MrFF assay was used to measure the growth inhibition at 12, 24, 48 h. Cell morphology was examined by the inverted microscope after treated for 24 h, and flow cytometry was used to evaluate the apoptosis, then we measured the ERS- related protein expressions of GRP78, GRP94, Perk, p-Perk, Chopand Caspase-4 by Western blot. RESULTS： ASMase activity continuously increased for VES （20 μg/mL） treatment, peaked at 3 h, then gradually decreased. ASMase activity of VES＋DES group remained at a low level. There was no significant change in control group and DES group throughout the tests. Cell proliferation in VES group and VES＋DES group was obviously inhibited after treatment for 12, 24 and 48 h （all P〈0.05） and showed a time-effect trend. Cell relative growth rates at different time points in VES＋DES group were significantly higher than those in VES group（all P〈0.05）. The number of dead cells obviously increased in VES group, and the number of dead cells in VES＋DES group decreased compared with VES group. Apoptosis rate evaluated by flow cytometry revealed that VES group （39.21% ＋ 1.90%） was significantly higher than the control group（3.91% ＋ 1.68%） and the DES group（4.07% ＋ 1.39%）（P〈0.05）, VES＋DES group （19.47%± 4.46%） was significantly lower than the VES group（P〈0.0