ApoG2对胃癌细胞SGC7901增殖和凋亡影响的观察

目的:研究Apogossypolone(ApoG2)对人胃癌细胞系SGC7901体外增殖和凋亡的作用。方法:采用MTT法检测浓度分别为3.125、6.250、12.500、25.000和50.000μmol/L的ApoG2作用于SGC7901细胞24、48和72h后对细胞生长的影响;MGG染色法观察10、20和30μmol/L ApoG2作用24h后细胞形态学的变化;流式细胞术检测10、20、30和40μmol/L ApoG2作用24h后细胞凋亡;RT-PCR方法检测10、20和30μmol/L ApoG2作用细胞24h后,SGC7901细胞中Bcl-2、Bax和NF-κB mRNA表达量的变化。结果:随着药物浓度的增大,ApoG2抑制胃癌细胞增殖的作用逐渐增强,呈剂量依赖性(P<0.05),24、48和72h的半数抑制浓度分别为32.58、25.11和14.16μmol/L;MGG染色可见,随着药物浓度的增大,细胞的生长变慢,胞质变疏松,细胞核深染,核质比例增大,出现典型的细胞死亡形态。流式细胞术检测细胞凋亡可见,经不同浓度的ApoG2处理24h后,SGC7901细胞发生凋亡,0μmol/L早期凋亡率为(1.92±0.55)%,晚期凋亡率为(2.80±0.86)%,10μmol/L早期凋亡率为(3.36±0.55)%,晚期凋亡率为(13.09±0.93)%,20μmol/L早期凋亡率为(5.07±0.70)%,晚期凋亡率为(16.48±1.03)%,30μmol/L早期凋亡率为(7.44±1.47)%,晚期凋亡率为(18.32±1.44)%,40μmol/L早期凋亡率为(7.88±1.22)%,晚期凋亡率为(25.49±1.59)%,随药物浓度的增加凋亡率明显增高,P值均<0.01;RT-PCR检测结果显示,药物干预后,胃癌细胞中Bcl-2和NF-κB的mRNA表达降低,而Bax的表达量升高,差异有统计学意义,P<0.05。结论:ApoG2在体外具有抑制胃癌细胞SGC7901的增殖并杀伤肿瘤细胞的作用。     

rAd-p53联合顺铂对胃癌细胞生长及 KAI1／CD82蛋白表达的影响

目的：观察rAd-p53、顺铂（ DDP）单独及联合作用于人胃癌SGC7901细胞后,对肿瘤细胞增殖凋亡情况、KAI1／CD82蛋白表达情况的影响。方法 rAd-p53、DDP单独及联合作用于胃癌SGC7901细胞株24、48、72 h后,CCK-8法测定SGC7901细胞体外增殖活性,流式细胞术检测细胞凋亡率,免疫组化法检测KAI1／CD82蛋白表达情况。结果rAd-p53、DDP单独及联合作用于SGC7901细胞后,细胞增殖被抑制,并呈剂量和时间依赖性,且两药联合组细胞增殖抑制率明显高于单用组,与阴性对照组相比差异均有统计学意义（ P＜0．05）;rAd-p53、DDP单独及两药联合作用SGC7901细胞48h后,细胞凋亡率为36．94％±0．78％、28．79％±2．37％,69．26％±0．63％;rAd-p53、DDP单独及两药联合均可上调KAI1／CD82蛋白表达,且两药联合组更明显。结论 rAd-p53、DDP单独及两药联合均可抑制胃癌SGC7901细胞的生长,诱导其凋亡,二者联合对胃癌细胞的抑制作用增强,rAd-p53可能通过上调KAI1／CD82的表达诱导SGC7901细胞凋亡,同时增强DDP的抗肿瘤作用。     

一种新型小分子化合物增强PC3细胞的辐射敏感性

目的 检测小分子化合物8-氧-8H-苊并[1,2-b]吡咯-9-腈的氨基取代衍生物（S1）对前列腺癌细胞系PC3辐射敏感性的作用。方法 采用流式细胞术分别检测不同剂量X射线和不同浓度S1化合物以及两者联合作用后,PC3细胞凋亡率的变化;采用Western blot方法检测S1作用后不同时间Bcl-2蛋白在PC3细胞中的表达变化。结果 X射线诱导PC3细胞凋亡具有剂量依赖特性,2Gy、5Gy X 射线照射未发现明显的凋亡,10Gy、20Gy照射组有明显的凋亡产生 (P<0.01);S1化合物能够显著诱导PC3细胞凋亡,并具有剂量依赖特性,其中5μmol/L和10μmol/L组凋亡率明显高于对照组（P<0.01）;S1作用后2~12h PC3细胞中Bcl-2蛋白表达逐渐减低;S1作用后,分别给予PC3细胞2Gy和8Gy X射线照射,细胞凋亡率明显高于单独照射组和单独S1作用组（P<0.01）。结论 S1能够增强PC3细胞对X射线的敏感性,可望为临床提高前列腺癌放疗效果,降低辐射损伤提供新的治疗方案。     

金雀异黄素协同TRAIL诱导乳腺癌MCF-7细胞凋亡作用的研究

目的：研究金雀异黄素是否协同TRAIL诱导乳腺癌MCF-7细胞发生凋亡, 并探讨其可能的内在机制。方法： 首先,MCF-7细胞分别经过1.25, 2.5, 5, 10, 20, 40 μg/mL金雀异黄素及1, 10, 100, 1 000 ng/mL TRAIL单独处理后, 应用MTT法检测MCF-7细胞增殖情况, 根据其结果选择终浓度为20 μg/mL金雀异黄素及100 ng/mL TRAIL作为后续试验的浓度; 接着, MCF-7细胞分为4组, 即对照组、 Gen组 （终浓度为20 μg/mL）、 TRAIL组 （终浓度为100 ng/mL） 及Gen+TRAIL组。细胞经不同处理后, 流式细胞仪检测细胞凋亡率; 免疫荧光法检测细胞凋亡中Caspase-3活性; 应用酶联免疫吸附方法检测细胞NF-кB含量。结果： 联合应用Gen后, 明显增强TRAIL对MCF-7细胞增殖的抑制 ［抑制率为 （63.78±2.61） %］, 并且促进TRAIL诱导细胞凋亡的发生 ［凋亡率为 （42.20±1.35） %］, 均分别高于相应的单独TRAIL处理组 （P<0.01）。另外, 经TRAIL单独处理的MCF-7细胞Caspase-3活性为17.324±0.880 μmol/L/hr/mg protein, NF-κB的含量为343.333±8.064 pg/mL; 而在联合应用Gen以后, Caspase-3活性增高 （44.000±0.445 μmol/L/hr/mg protein）, 同时NF-κB的合成受到抑制 （177.453±25.389 pg/mL）, 与相应单独用药组比较差异具有统计学意义（P<0.01）。结论： 金雀异黄素可协同TRAIL诱导乳腺癌MCF-7细胞发生凋亡、 增加乳腺癌细胞对TRAIL的敏感性, 其可能的机制是Gen协同TRAIL激活了细胞凋亡过程中Caspase-3, 并进一步抑制了NF-κB的合成, 从而最终导致乳腺癌MCF-7细胞凋亡的发生。     

GNA对人宫颈癌细胞增殖抑制、凋亡、迁移及细胞周期分布的影响

目的:研究新藤黄酸（gambogic acid,GNA）对人宫颈癌细胞的增殖抑制、凋亡、迁移以及细胞周期分布的影响。方法:将人宫颈癌Hela细胞分为空白对照组、25 μmol/L浓度组、50 μmol/L浓度组和100 μmol/L浓度组,进行细胞培养后采用MTT法和流式细胞术检测细胞24 h、48 h和72 h时间段的增殖抑制和凋亡情况,Transwell实验检测细胞迁移情况,流式细胞仪检测细胞周期分布,Western blot检测Bcl-2、Bax、E-cadherin和NF-κB蛋白相对表达量。结果:25 μmol/L浓度组、50 μmol/L浓度组和100 μmol/L浓度组对宫颈癌细胞的抑制增殖率在不同时间段均显著高于空白对照组,增殖抑制率随着浓度和时间的增加而升高（P＜0.05）;25 μmol/L浓度组、50 μmol/L浓度组和100 μmol/L浓度组宫颈癌细胞凋亡率在各时间段均显著高于空白对照组,凋亡率随着浓度和时间的增加而升高（P＜0.05）;25 μmol/L浓度组、50 μmol/L浓度组和100 μmol/L浓度组宫颈癌细胞的细胞迁移数量相比对照组均显著减少,细胞迁移数量随着浓度的增加而减少（P＜0.05）;GNA浓度越高,处于G0/G1期的细胞比例越高,处于G2/M和S期的细胞比例越低;25 μmol/L浓度组、50 μmol/L浓度组和100 μmol/L浓度组Bcl-2、NF-κB均低于空白对照组（P＜0.05）;25 μmol/L浓度组、50 μmol/L浓度组和100 μmol/L浓度组Bax、E-cadherin表达均高于空白对照组。结论:GNA能够促进宫颈癌细胞的凋亡,抑制细胞的增殖和迁移能力,通过改变癌细胞的周期分布降低癌细胞的增长,其能力呈现浓度依赖性。     

β-榄香烯联合紫杉醇诱导胃癌细胞凋亡的体外研究

目的:观察联合β-榄香烯和紫杉醇对诱导胃癌细胞凋亡以及对凋亡相关蛋白表达的影响。方法:β-榄香烯和紫杉醇单独及联合作用于胃癌SGC7901细胞,MTT法检测细胞增殖抑制率,流式细胞术检测细胞凋亡,免疫印记检测凋亡相关蛋白的表达。结果:单药β-榄香烯作用SGC7901细胞24、48和72小时的IC50值分别为(52.56±6.26)、(28.69±2.36)和(13.33±0.64)μg/ml。单药紫杉醇作用24、48和72小时的IC50值为(7.23±1.98)、(1.72±0.76)和(0.22±0.19)μg/ml。选用低浓度药物(10μg/ml的β-榄香烯和2μg/ml的紫杉醇)联合处理SGC7901细胞24小时,与相应浓度β-榄香烯、紫杉醇单药组相比,两药联合组对SGC7901的增殖抑制率显著增强(P<0.05),细胞凋亡率显著提高。进一步研究发现β-榄香烯和紫杉醇联合后,诱导了PARP裂解以及显著下调了Bcl-2与Bax的比值。结论:β-榄香烯和紫杉醇联合用药后,可能通过下调Bcl-2与Bax的比值诱导PARP裂解,进而抑制胃癌细胞增殖和诱导细胞凋亡。     

YKL-40调控子宫内膜癌顺铂化疗耐药性的实验研究

目的 探讨沉默甲壳质酶蛋白 40（YKL-40）表达对子宫内膜癌顺铂（DDP）耐药细胞株Ishikawa／DDP的影响。方法 采用DDP长期浓度梯度递增法体外建立Ishikawa／DDP耐药细胞株，并检测多药耐药相关基因（MDR1）和Bcl-2、Bax和caspase-3的表达，采用MTT法检测Ishikawa细胞和Ishikawa／DDP细胞的增殖活性，计算半数抑制浓度（IC50）。Ishikawa／DDP分别转染NC阴性序列（NC组）和siRNA YKL-40（si-YKL-40组），另设未转染细胞作为对照组，采用MTT法、划痕实验和Annexin V／PE双染法检测DDP对si-YKL-40组Ishikawa／DDP细胞增殖、迁移能力和凋亡的影响。结果 体外成功建立Ishikawa／DDP耐药细胞株，3.125、6.25、12.5、25、50、100 μmol／L DDP对Ishikawa／DDP细胞的增殖抑制率分别为（6.93±2.45）％、（8.14±4.50）％、（11.37±4.62）％、（15.18±3.97）％、（26.29±5.08）％、（41.32±7.64）％，明显低于Ishikawa细胞（P＜0.05）。DDP对Ishikawa和Ishikawa／DDP的IC50分别为14.58 μmol／L和116.70 μmol／L，Ishikawa／DDP的耐药指数为8.004。QPCR检测结果显示，Ishikawa细胞YKL-40、MDR1、Bcl-2、Bax、caspase-3 mRNA表达量分别为0.82±0.15、0.43±0.11、1.05±0.23、1.17±0.20、0.96±0.18，而Ishikawa／DDP细胞分别为1.87±0.40、2.34±0.46、1.52±0.28、0.72±0.21、0.49±0.17，差异有统计学意义（P＜0.05）。3.125、6.25、12.5、25、50、100 μmol／L DDP对si-YKL-40组细胞的增殖抑制率分别为（10.95±2.74）％、（18.73±5.30）％、（32.79±5.47）％、（52.28±6.58）％、（61.73±5.26）％、（65.45±7.33）％，明显高于对照组和NC组，差异有统计学意义（P＜0.05）。DDP对si-YKL-40组细胞的IC50为22.19 μmol／L。与对照组和NC组比较， si-YKL-40组细胞凋亡率升高、愈合率下降；YKL-40、MDR1、Bcl-2蛋白表达量下调， Bax和caspase-3蛋白表达量上调，差异有统计学意义（P＜0.05）。结论 沉默YKL-40可显著提高Ishikawa／DDP细胞株对DDP的敏感性，并促进细胞凋亡，其具体机制可能与下调MDR1、Bcl-2表达，及上调Bax和caspase-3表达有关。     

熊果酸抑制胃癌细胞SGC7901增殖和诱导细胞凋亡的机制

背景与目的:研究表明熊果酸(ursolicacid,UA)可抑制多种肿瘤细胞的增殖并诱导凋亡,但目前有关UA作用于胃癌细胞的报道较为少见。环氧合酶-2(cyclooxygenase-2,COX-2)在多种癌前病变及癌组织中高表达。本研究旨在探讨熊果酸抑制人胃癌细胞SGC7901增殖和诱导凋亡的机制。方法:MTT法检测0、10、20、30、40!mol/LUA作用不同时间对SGC7901细胞增殖的影响;荧光染料Hoechst33258染色观察不同浓度UA作用24h细胞凋亡情况;流式细胞仪检测细胞周期变化及凋亡率;Westernblot法检测COX-2蛋白以及凋亡相关蛋白Bcl-2、Bax表达。放射免疫分析法测定COX-2催化产物前列腺素E2(prostaglandinE2,PGE2)。结果:20～40!mol/LUA可抑制SGC7901细胞的增殖,并呈浓度和时间依赖性,作用12、24、36、48h的半数抑制浓度(IC50)分别为(57.50±1.18)!mol/L、(34.28±2.05)!mol/L、(27.54±1.11)!mol/L、(24.83±1.02)!mol/L;20～40!mol/LUA作用24h后,SGC7901细胞被阻滞于G0/G1期,细胞凋亡率分别为(9.10±2.39)%、(26.30±1.25)%、(35.20±2.26)%;同时COX-2蛋白表达及其催化生成产物PGE2浓度下降,凋亡相关蛋白Bcl-2表达减少,Bax无明显变化。结论:熊果酸对SGC7901细胞具有增殖抑制及诱导凋亡作用,其机制可能与阻滞细胞周期、抑制COX-2表达进而减少PGE2生成以及下调凋亡相关蛋白Bcl-2表达有关。     

全反式维甲酸增强顺铂对A549 细胞化疗敏感性及对Survivin mRNA 和COX-2 mRNA表达的影响

目的:体外观察全反式维甲酸（all-trans retinoic acid ,ATRA）增强顺铂（cisplatin,DDP ）对人类非小细胞肺癌细胞株A549 细胞的增殖抑制及对凋亡抑制蛋白Survivin mRNA和环氧化酶-2（cyclooxygenase- 2,COX-2）mRNA 表达的影响。方法:应用不同浓度组DDP（0.5、5、50mg/L）、ATRA（0.1、1、10μ mol/L）以及联合用药组（ATRA 1 μ mol/L,DDP 5mg/L）,处理肺腺癌细胞株A549 细胞,采用四甲基偶氮唑盐（MTT）比色法观察不同浓度DDP 组、不同浓度ATRA 组及联合用药组对A549 细胞生长的影响;应用实时荧光定量聚合酶链反应（real-time polymerase chain reaction ,RT-PCR）检测DDP 组、ATRA 组及联合用药组处理前后A549 细胞中Survivin mRNA和COX-2 mRNA 表达变化;应用流式细胞术观察DDP 组、ATRA 组及联合用药组处理前后细胞凋亡率。结果:与空白对照组相比,单独应用DDP 、ATRA 处理A549 细胞均诱导细胞凋亡,且呈浓度依赖性。与单独应用DDP 的作用相比,联合用药组可更显著抑制A549 的增殖,增加细胞的凋亡率（P<0.05）,并增强对A549 细胞Survivin mRNA和COX-2 mRNA表达的抑制作用（P<0.05）;并且,流式细胞术测定结果显示联合用药组的早期凋亡率（7.37± 3.83）% 、中晚期凋亡率（34.37±2.08）% 、继发性坏死率（7.44± 0.46）% 均较单独应用DDP 组高（3.55± 0.75）% 、（6.62± 0.33）% 、（3.03± 0.05）% ,P 均<0.05。结论:全反式维甲酸能够明显提高非小细胞肺癌对顺铂的敏感性,其机制可能与抑制非小细胞肺癌细胞Survivin mRNA和COX-2 mRNA 的表达有关。      

Reversion of multidrug resistance by SKI-II in SGC7901/DDP cells and exploration of underlying mechanisms

In order to investigate whether SKI-II could reverse drug resistance and its possible mechanisms, we treated SGC7901/DDP cells with SKI-II or SKI-II in combination with DDP. Then cell growth, apoptosis, micro- morphological changes, and expression of SphK1, P-gp, NF-kB, Bcl-2 and Bax were assessed by MTT assay, flow cytometry, electron microscopy, immunocytochemistry and Western blot assay respectively. SGC7901/DDP cells were insensitive to cisplatin 2.5 mg/L, but when pretreated with SKI-II, their proliferation was inhibited by cisplatin 2.5mg/L significantly, the inhibition rate increasing with time and dose. The apoptosis rate was also significantly elevated. Expression of SphK1 and P-gp was decreased significantly, Pearson correlation analysis showing significant correlation between the two (r=0.595, P<0.01). Expression of NF-kB and Bcl-2 was decreased significantly, while that of Bax was increased, compared to the control group. There were significant correlations between SphK1 and NF-kB(r=0.723, P<0.01), and NF-kB and Bcl-2(r=0.768, P<0.01). All these data indicated that SKI-II could reverse drug resistance of SGC7901/DDP to cisplatin by down-regulating expression of P-gp and up-regulating apoptosis through down-regulation of SphK1. The increased apoptotic sensitivity of SGC7901/ DDP to cisplatin was due to the decreasing proportion of Bcl-2/Bax via down-regulating NF-kB.     

Combinational effect of PPARγ agonist and RXR agonist on the growth of SGC7901 gastric carcinoma cells in vitro

In order to investigate the inhibitory effects and mechanisms of troglitazone (TGZ), a peroxisome proliferator-activated receptor γ (PPARγ) agonist, and retinoid X receptor (RXR) agonist (9-cis-retinoic acid (RA)) on gastric carcinoma cells SGC7901, SGC7901 cells were treated with TGZ and 9-cis-RA, respectively, or in combination. Then, the cell growth, apoptosis, morphological changes, and the expression of PPARγ, RXRγ, Bcl-2, and Bax were detected by MTT assay, flow cytometry, HE staining, immunocytochemistry staining, and Western blot assay, respectively. Our results showed that the growth of SGC7901 cells was inhibited and the cells got sparser at the concentrations of 50 μmol/L TGZ, 20 μmol/L 9-cis-RA, or combination of TGZ (25 μmol/L) and 9-cis-RA (10 μmol/L). Immunocytochemistry and Western blot showed that after 72 h, the expression of PPARγ, RXRγ, and Bax were upregulated; Bcl-2 was downregulated compared with the negative control group. These data indicated that PPARγ agonist and RXR agonist could inhibit the proliferation of SGC7901 cells via inducing the apoptosis, which involved the increase in the level of Bax/Bcl-2. The combination of RXR agonist and PPARγ agonist could induce the maximal inhibitory effects on tumor growth and apoptosis via promoting the formation of RXR/PPARγ heterodimer.     

A Sphingosine Kinase-1 Inhibitor,SKI-II,Induces Growth Inhibition and Apoptosis in Human Gastric Cancer Cells

SKI-II has been reported as an inhibitor of sphingosine kinase 1 and has been extensively used to prove theinvolvement of sphingosine kinase and sphingosine-1-phosphate (Sphk1) in cellular processes. In the currentstudy, we investigated the effects of SKI-II and its potential mechanisms in human gastric cancer SGC7901cells. After treatment with SKI-II, cell growth, cell cycle distribution, apoptosis, expression of Sphk1, NF-κB,Bcl-2, Bax and p27 were assessed by MTT assay, flow cytometry, electron microscopy, immunocytochemistryand Western-blot assay, respectively. Our results showed that SKI-II markedly inhibited SGC7901 cell survivalin a dose-dependent manner, reduced cell proliferation with accumulation of cells in the G0/G1 phase andinduced apoptosis in the tumor cells. Furthermore, Western blotting and immunocytochemistry showed that theexpression of p27 and Bax was increased significantly, but the expression of NF-κB, Bcl-2 and Sphk1 decreasedby different degrees. These results indicate that SKI-II induced cell growth arrest and apoptosis. The increasedapoptotic sensitivity of SGC7901 was correlated with NF-κB or Bcl-2/Bax activation.     

The Effect of Nimesulide on the Expression of NF-κB, Bcl-2 and Bax in the Human Gastric Cancer SGC-7901 Cell Line

OBJECTIVE To investigate whether nimesulide can suppress tumor growth and induce apoptosis in SGC-7901 gastric cancer cells and to explore the molecular mechanism involved.METHODS SGC-7901 cells were cultured in RPMI 1640 medium containing different concentrations of nimesulide (0,12.5, 50, 100, 200, 400 μmol/L). The MTT assay, morphological observation, electron microscopy (EM), immunohistochemical analysis and Western blot analysis were employed to investigate the effects of nimesulide on the SGC-7901 cells and to explore possible related molecular mechanisms.RESULTS Nimesulide inhibited the growth of SGC-7901 cells and elicited typical apoptotic morphologic changes. Nimesulide also decreased NF-κB and Bcl-2 expression, but increased the level of the Bax protein.The positive rate of Bcl-2 protein expression at 0, 50, 100 and 200 μmol/L of nimesulide was 58.3±14.0%, 50.2±9.9%, 32.8±5.0% and 22.7± 5.5% respectively based on immunohistochemical staining. The positive rate of Bax protein expression was 22.0±5.7%, 29.2±6.5%, 42.7±5.9% and 74.5±9.1% and the NF-κB expression was 74.2±10.9%, 61.8±7.6%,36.7±10.9% and 17.5±12.3%, Significant differences were found between 0 μmol/L and 100 μmol/L and 200 μmol/L. Western blot analysis also showed that the expression of NF-κB was decreased.CONCLUSION Nimesulide suppresses tumor growth and induces apoptosis by inhibiting NF-κB expression, which may be related to the overexpression of Bax relative to Bcl-2 expression.     

Autophagy Inhibition Sensitizes Cisplatin Cytotoxicity in Human Gastric Cancer Cell Line Sgc7901

We aimed to investigate the mechanism and effects of autophagy on cisplatin (DDP)-induced apoptosis inhuman gastric cancer cell line SGC7901. After SGC7901 cells were treated with DDP and/or chloroquine, cellproliferation was measured using MTT assay; cell apoptosis was determined by flow cytometry; autophagy andapotosis-related proteins expression were detected by Western blot; and quantitative analysis of autophagy aftermonodansylcadaverine (MDC) staining was performed using fluorescence microscopy. We found after treatmentwith 5 mg/L DDP for 24 h, the rates of cell apoptosis were (21.07±2.12)%. Autophagy, characterized by an increasein the number of autophagic vesicles and the level of LC3-II protein was observed in cells treated with DDP.After inhibition of autophagy by chloroquine, the rates of cell apoptosis were increased to (30.16±3.54)%, andthe level of Caspase-3 and P53 protein were increased, and Bcl-2 protein was decreased. Therefore, autophagyprotects human gastric cancer cell line SGC7901 against DDP-induced apoptosis, inhibition of autophagy canpromote apoptosis, and combination therapy with DDP and chloroquine may be a promising therapeutic strategyfor gastric cancer.     

As2S2对人卵巢癌耐药株C13K/DDP细胞增殖和凋亡的作用

 目的：研究As₂S₂对卵巢癌耐药株C13K/DDP细胞增殖抑制和诱导凋亡的作用。方法：以 不同浓度(4、6、8、10μmol/L)的As2S2,分三个时间点(24、48、72h)干预C13K/DDP细胞,采用 四甲基偶氮唑蓝(MTT)法检测As2S2对C13K/DDP细胞的增殖抑制率;流式细胞仪(FCM)检测细胞凋 亡率;Western blot检测BCL-2、BAX、AKT的表达。结果：MTT结果显示不同浓度(4、6、8、10 μmol/L)的As₂S₂作用C13K/DDP细胞后,与DDP组相比其增殖受到抑制,作用呈明显的时效和量效 关系,差异有统计学意义(P<0.01);流式细胞仪的结果显示6、8μmol/L As₂S₂诱导细胞的24h凋 亡率分别为(16.05 ±2）%、( 22.30±3）%,DDP组为(9.45 ±2)%,对照组为( 7.82±1.2)% ;6、8μmol/L As₂S₂诱导细胞48h凋亡率分别为(28.94 ±1.8）%、(37.85 ±3）%,DDP组为 （14.74±3.2）%,对照组为( 9.80±2.6）%,各组比较,差异有统计学意义(P<0.05);Western blot结果显示BCL-2、AKT表达下调,BAX表达明显上调。结论：As₂S₂对人卵巢癌耐药株 C13K/DDP细胞具有增殖抑制和诱导凋亡的作用,可能与BCL-2下调、BAX上调或AKT下调有关。     

氯喹促进顺铂诱导胃癌SGC7901细胞凋亡及其机制

  目的  研究自噬对顺铂（cisplatin,DDP）诱导的胃癌SGC7901细胞凋亡的影响,并初步探讨其可能机制。  方法  DDP和（或）氯喹处理胃癌SGC7901细胞,MTT法检测细胞增殖,流式细胞术检测细胞凋亡,MDC染色后荧光显微镜观察自噬囊泡,West. ern Blot检测自噬和凋亡相关蛋白。  结果  5 mg/L的顺铂作用于胃癌SGC7901细胞24 h,细胞凋亡率为21.07%±2.12%,同时观测到自噬囊泡增多和LC3-Ⅱ蛋白表达升高;氯喹特异性抑制自噬活性后,提高了顺铂诱导的细胞凋亡率（30.16%±3.54%,P < 0.05）;检测到凋亡相关蛋白Caspase-3和P53表达增加,Bcl-2蛋白表达下降。  结论  自噬在顺铂诱导胃癌SGC7901细胞凋亡的过程中起保护作用,氯喹抑制自噬后,可能通过激活P53蛋白及灭活Bcl-2蛋白的表达来促进细胞凋亡,联合应用顺铂和氯喹有望成为胃癌治疗的新策略。      

RAD001 can reverse drug resistance of SGC7901/DDP cells

To investigate the role of RAD001 in the reversing of drug resistance of SGC7901/DDP, we cultured SGC7901/DDP cells with different groups of drugs (RAD001, cisplatin (DDP) alone, or the combination of RAD001 and DDP); after that, we detected the drug sensitivity, cell apoptosis, and levels of P-gp, MRP1, and survivin in the cells of SGC7901/DDP by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphe-nyltetrazolium bromide) assay, flow cytometry, immunohistochemical analysis, and Western blot analysis. There was no significant difference between DDP 2.5-mg/L group and negative control group. When the cells were pretreated with RAD001 2.5, 5 nmol/L, the proliferation of SGC7901/DDP cells was inhibited by DDP 2.5 mg/L significantly, compared to negative control group, DDP 2.5-mg/L group, and RAD001 2.5, 5-nmol/L group, respectively (P?P?RAD001 2.5 nmol/L did not induce apoptosis of SGC7901/DDP cells alone (P?>?0.05). When SGC7901/DDP cells were pretreated with RAD001 2.5 nmol/L, DDP 2.5 mg/L increased the apoptosis rate significantly compared to groups of control and DDP 2.5 mg/L alone (P?5, Fig. 2) and Western blot analysis (Fig. 3) indicated that when SGC7901/DDP cells were pretreated with RAD001 2.5 nmol/L, the expression of P-gp, MRP1, and survivin decreased by different degrees. Our results have confirmed that RAD001 in combination with DDP could overcome chemoresistance of SGC7901/DDP cells by decreasing the levels of P-gp, MRP1, and survivin through the mTOR pathway.     

抑制核转录因子活化逆转胃癌细胞株耐药性研究

目的　探讨核转录因子（ＮＦ-κＢ）的活化在胃癌细胞株耐药性机制中的作用。方法　用阿霉素（ＡＤＭ）诱导培养胃癌细胞耐药亚株ＳＧＣ７９０１／ＡＤＭ,ＭＴＴ法检测ＡＤＭ对胃癌细胞株的细胞毒作用,流式细胞仪检测胃癌细胞株凋亡率的变化,免疫细胞化学染色法检测胃癌细胞株ＮＦ κＢ的活化情况。吡咯烷二硫代氨基甲酸脂（ＰＴＤＣ）抑制ＮＦ-κＢ活化,并检测ＡＤＭ对胃癌细胞株的细胞毒作用。结果　ＳＧＣ７９０１／ＡＤＭ的ＩＣ_５０为２.６３μｇ／ｍｌ,ＳＧＣ７９０１的ＩＣ_５０为０.２９μｇ／ｍｌ,ＳＧＣ７９０１／ＡＤＭ相对耐药度较亲本细胞提高了８.９倍。４８ｈ内ＳＧＣ７９０１和ＳＧＣ７９０１／ＡＤＭ胃癌细胞株发生的凋亡率随ＡＤＭ浓度增加而升高,随着作用时间延长而升高;１０μｇ／ｍｌＡＤＭ分别作用４８ｈ时ＳＧＣ７９０１和ＳＧＣ７９０１／ＡＤＭ胃癌细胞株发生的凋亡率为（４８.５３±１.０２）％和（１７.５３±１.０２）％。免疫细胞化学染色显示ＡＤＭ作用ＳＧＣ７９０１／ＡＤＭ胃癌细胞９ｈ后可检测到ＮＦ-κＢ核移位,最高达到６５％;ＡＤＭ作用ＳＧＣ７９０１胃癌细胞株１８ｈ后可检测到少量ＮＦ-κＢ核移位;所有的细胞株ＮＦ-κＢ核移位均于２４ｈ后减弱;ＰＴＤＣ抑制核转录因子活化后ＡＤＭ细胞毒效应增强（Ｐ＜０.０５）。结论　ＮＦ-κＢ活化所导致的胃癌细胞株凋亡率下降在胃癌细胞株耐药性机制中发挥一定作用,抑制ＮＦ-κＢ活化后可以逆转胃癌细胞株对ＡＤＭ耐药性。