【作者】 王亚楠；

【导师】 樊青霞；

【作者基本信息】 郑州大学 ， 肿瘤学， 2014， 硕士

【摘要】 背景胃癌是目前世界上第四大最常见的癌症，新近诊断的肿瘤病例中8%为胃恶性肿瘤。尽管在近数十年以来手术方法，化疗和支持治疗得到提高，但其总体生存时间并没有显著提高。胃癌已成为世界范围内癌症致死病例中的第二大死因（738,000死亡病例，约占总数9.7%）。现胃癌的发病原因尚不明确，由于早期阶段无明显症状，因此约有65%的胃癌病人在确诊时已出现局部或远处转移，从而不能进行手术切除，这使得治疗变得困难。转移性、进展性胃癌患者的5年生存率很低，仅有5%-15%。对于胃癌来说，化疗是除手术切除外的一种重要的治疗手段，但由于肿瘤细胞化疗耐药性的出现，使得这一治疗的有效性下降。寻找肿瘤治疗新的靶点或化疗增敏药物，已成为胃癌临床治疗中亟待解决的问题。目的探讨鞘氨醇激酶-1（SphK1）抑制剂N,N-二甲基鞘氨醇（DMS）与5-氟尿嘧啶(5-FU)联用对胃癌MGC-803细胞增殖和凋亡的影响及其可能的机制。方法体外常规培养胃癌MGC-803细胞，采用MTT比色法来检测不同浓度DMS、5-FU单药和（或）两药联合对MGC-803细胞增殖的抑制情况；流式细胞术检测其细胞周期分布和凋亡率；Western blot法测定药物作用后MGC-803细胞中SphK1、TS、DPD、NF-κB p65和bcl-2蛋白表达的变化。结果1.DMS和5-FU单药及联合作用对MGC-803细胞增殖抑制的影响（1）不同浓度DMS(分别为1.25、2.5、5、10和20μmol/L)、5-FU（100μmol/L）均能够抑制胃癌MGC-803细胞增殖。且随着DMS药物浓度的增加，作用时间的延长，其增殖抑制率增加，呈现出浓度-时间依赖性。（2）与DMS、5-FU单药组相比，两药联合对胃癌MGC-803细胞的增殖抑制作用显著增加，差异具有统计学意义（P＜0.05）。2.DMS和5-FU单药及联合作用对MGC-803细胞周期分布的影响与空白对照组比较，DMS（5μmol/L）单药作用胃癌MGC-803细胞后其细胞周期的各期比例无明显变化（P＞0.05）；5-FU（100μmol/L）组细胞G0/G1期细胞比例增加至（61.71±1.33）%，S期细胞比例减少至（28.06±1.09）%（P＜0.05）；联合组G0/G1期细胞比例进一步增加至（63.34±1.64）%，但与5-FU组比较，其差异无明显统计学意义（P＞0.05）。3.DMS和5-FU单药及联合作用对MGC-803细胞凋亡的影响（1）1.25、2.5、5.0和10μmol/L DMS单药分别作用于MGC-803细胞48h后，细胞凋亡率分别为（10.27±0.91）％、（14.77±1.35）％、（20.07±1.56）％和（34.53±1.92）％，而对照组细胞凋亡率为（0.53±0.06）％。统计分析后显示，随着DMS浓度的增加，MGC-803细胞凋亡率也逐渐上升，呈明显的浓度依赖性，与空白对照组比较，差异具有统计学意义（P＜0.05）。（2）DMS（5μmol/L）、5-FU（100μmol/L）单药及两药联合作用于MGC-803细胞48h后，MGC-803细胞的凋亡率分别为（20.07±1.56）％、（21.63±1.80）％和（25.60±1.28）％。统计分析后显示，DMS与5-FU联合应用时可明显增加MGC-803细胞凋亡率，高于DMS组、5-FU组和对照组（P＜0.05）。4. DMS和5-FU单药及联合作用对MGC-803细胞SphK1、TS、DPD、NF-κB p65和bcl-2蛋白表达的影响Western-blot检测结果显示，与对照组比较，DMS单药组和联合组SphK1、NF-κB p65、bcl-2蛋白表达水平均明显下降（P＜0.05），且与DMS药物浓度相关；而联合组SphK1、NF-κB p65、bcl-2蛋白表达水平均明显低于DMS单药组（P＜0.05）；而各组细胞TS、DPD蛋白的表达无明显变化（P＞0.05）。结论SphK1抑制剂DMS能够抑制胃癌MGC-803细胞的增殖并诱导凋亡，与5-FU联用后其增殖抑制及凋亡诱导作用增强，其机制可能与SphK1、NF-κB p65、bcl-2蛋白表达下调相关。更多还原

【Abstract】 BackgroundGastric cancer is currently the fourth most common cancer in the world andnewly diagnosed cancer cases in8%of gastric malignancy. Although the operationmethod, the treatment of chemotherapy and support improved in recent decades,overall survival was not significantly increased.Gastric cancer is the second leadingcause of cancer death in the word (738,000deaths, accounting for9.7%of total). Thepathogenesis of gastric cancer is not clear. Since in the early stages of gastric cancerhave no obvious symptoms, about65%of gastric cancer patients have local or distantmetastases at the time of diagnosis, so they lost the opportunities of surgery, makingtreatment difficult. The5-year survival rate of patients with metastatic or advancedgastric cancer is very low, only5%-15%. Chemotherapy is an important therapeuticmodality for gastric cancer besides surgical resection,although the success rate of thistreatment is limited because of chemoresistance. Looking for a new target orchemosensitizer in the clinical treatment of gastric cancer is urgently needed. ObjectivesTo investigate the effects of sphingosine kinase1(SphK1) inhibitor N,N-dimethylsphingosine (DMS) combined with5-fluorouracil (5-FU) on theproliferation and apoptosis of gastric cancer MGC-803cells and to explore thepossible mechanisms involved.MethodsMGC-803cells were cultured in vitro. The effects of DMS and5-FU on cellproliferation, apoptosis, and cell cycle distribution of MGC-803cells were detectedby MTT assay and flow cytometer (FCM), respectively. The expressions of SphK1,TS, DPD, NF-κB p65and bcl-2proteins were detected by Western blot.ResultsDifferent concentrations of DMS or5-FU alone or in combination couldobviously inhibit the proliferation of MGC-803cells in a dose-dependent andtime-dependent manners (P＜0.05). And the proliferation inhibition rate of MGC-803cells in the combination group was significantly higher than that in the single druggroups (P＜0.05). Treatment of MGC-803cells with DMS did not affect the cellcycle distribution (P＞0.05). As compared with the cells without drug treatment,DMS or5-FU alone could obviously increase the apoptosis rate of MGC-803cells (P＜0.05); the apoptosis rate in the combination group was significantly higher thanthat in the single-drug groups (P＜0.05). The expression levels of SphK1, NF-κB p65and bcl-2proteins were down-regulated with the treatment of DMS alone or incombination, whereas those of TS and DPD were not affected.ConclusionDMS can inhibit the proliferation and induce apoptosis of gastric cancerMGC-803cells in vitro. It shows a good synergetic effect in combination with5-FU,probably by down-regulating the expressions of SphK1, NF-κB p65and bcl-2 proteins.更多还原