【作者】 陈冬梅；

【导师】 李建英；

【作者基本信息】 福建医科大学 ， 内科学， 2010， 硕士

【摘要】 目的:脂氧合酶对肿瘤的作用已成为肿瘤研究领域的热点之一,本研究旨在研究15-LOX-1基因抑制人胃癌细胞AGS增殖,诱导其凋亡的作用机制。方法:通过脂质体介导瞬时转染15-LOX-1基因于胃癌细胞AGS中,以转染空质粒PCDNA3.1的AGS细胞及未转染质粒的AGS细胞为对照,RT-PCR法检测15-LOX-1和PPARγmRNA表达;Western blot法检测15-LOX-1、PPARγ、P27/Skp2、Bax/Bcl-2蛋白表达;四甲基偶氮唑盐法(MTT)检测15-LOX-1和GW9662对胃癌细胞AGS增殖水平的影响。结果:(1)胃癌细胞AGS中未检测到15-LOX-1mRNA和蛋白的表达,转染后的AGS表达有15-LOX-1的mRNA及蛋白。(2)转染15-LOX-1后细胞周期抑制因子P27表达较未转染和转染空载质粒组显著上调,而S相激酶相关蛋白2(Skp2)表达下降(P<0.05)。(3)转染48小时后,AGS/15-LOX-1组细胞存活率为58.5%,显著低于AGS及AGS/PCDNA3.1组(P<0.05)。AGS/15-LOX-1组细胞加入10uM GW9662作用48小时后,细胞存活率为76.2%,较GW9662作用前显著升高(P<0.05)。(4) AGS、AGS/15-LOX-1、AGS/PCDNA3.1组均能检测到PPARγmRNA和蛋白的表达,且AGS/15-LOX-1组PPARγ表达量较其他两组显著增多(P<0.05),10uM GW9662能够下调AGS/15-LOX-1组PPARγ表达。(5) AGS/15-LOX-1组Bax蛋白的表达较AGS和AGS/PCDNA3.1组上调,而Bcl-2蛋白的表达量下降(P<0.05),加入GW9662作用48h后,AGS/15-LOX-1组Bax/Bcl-2蛋白的表达量与AGS和AGS/PCDNA3.1组无显著差别(P>0.05)。结论:15-LOX-1可能经由PPARγ途径,通过调节细胞周期蛋白Skp2和P27的表达,使细胞周期阻滞于G0/G1期,并通过调节下游凋亡相关基因Bax/Bcl-2的表达,抑制细胞增殖,促进其凋亡。更多还原

【Abstract】 Objective: The study was to investigate the mechanism of proliferation inhibition and apoptosis induction by 15-lipoxygenase-1(15-LOX-1) gene on gastric cancer cell AGS.Methods: AGS cells were divided into three groups: AGS/15-LOX-1 group transfected with PCDNA3.1/15-LOX-1,vector control group transfected with PCDNA3.1 and blank control group without transfection.Plasmids of PCDNA3.1/15-LOX-1 encoding 15-LOX-1 gene were transiently transfected into AGS cells.The expression of 15-LOX-1 and PPARγat mRNA level were measured by reverse transcription polymerase chain reaction(RT-PCR).The expression of 15-LOX-1、PPARγ、Skp2/P27、Bax/Bcl-2 at protein level were measured by western blot.The proliferation of four groups cells(AGS/15-LOX-1、AGS、AGS/15-LOX-1 add with GW9662、AGS add with GW9662) were assessed by thiazolyl blue tetrazolium bromide(MTT) assay respectively.Results: (1) 15-LOX-1 mRNA and protein were expressed in AGS/15-LOX-1 cells compared with control groups detected by RT-PCR and western blot.(2) P27 which was expressed from AGS cells transfected with PCDNA3.1/15-LOX-1 is up-regulated, meanwhile, S-phase Kinase-associated Protein 2 (Skp2) is down-regulated compared with control groups(P<0.05).(3)There was a significant decline in cell proliferation ability of AGS/15-LOX-1 in comparision with control groups revealed by MTT assay(P<0.05).Cell survival rates were 58.5%,48h after transfection.After added with 10uM GW9662, cell proliferation ability of AGS/15-LOX-1 were 76.2%,48h after intervention.(4)PPARγmRNA and protein were expressed in AGS/15-LOX-1 cells、AGS and AGS/pcDNA3.1 cells detected by RT-PCR and western blot.Also,in AGS/15-LOX-1 cells,the expression of PPARγ was significantly higher than that in AGS and AGS/pcDNA3.1 cells(P<0.05).(5)Bax which was expressed from AGS cells transfected with 15-LOX-1 is up-regulated, meanwhile, Bcl-2 is down-regulated compared with control groups(P<0.05).But 48h after intervent with GW9662, western blot confirmed that there was no statistically significant differences in comparision with control groups(p>0.05).Conclusion:The expression of 15-LOX-1 could effectively regulate the expression of Skp2 and P27 to resort gastric carcinoma cell in period G0/G1 and inhibit carcinoma cell proliferation.The expression of 15-LOX-1 could up-regulated expression of bax and low-regulated expression of Bcl-2 through PPARγ,which may be the mechanism of 15-LOX-1 gene induce the apoptosis of human gastric cancer AGS cells.更多还原