摘要
目的:研究缬草波春诱导胃癌细胞凋亡,探讨其诱导凋亡与半胱氨酸酶(Caspase)及生存素(Survivin)mRNA、P53蛋白、Survivin蛋白表达的关系.方法:以100mg/L的缬草波春作用于加Caspase-3抑制剂、Caspase-8抑制剂、Caspase-9抑制剂和未加Caspases抑制剂培养的MKN-45细胞24,48和72h,用流式细胞仪分别检测细胞凋亡率;不同浓度的缬草波春(5,10,25,50,100mg/L)作用MKN-45细胞不同时间(24,48,72h)后,用tripure提取液提取细胞RNA,用RT-PCR法,检测SurvivinmRNA的表达.不同浓度缬草波春(50和100mg/L)作用MKN-45胃癌细胞株24h后,用免疫组化的方法,检测P53蛋白和Survivin蛋白的表达.结果:单用Caspase抑制剂组,作用24,48和72h对MKN-45细胞凋亡率无明显影响,与对照组比较差异无显著意义.Caspase-3抑制剂、Caspase-9抑制剂与缬草波春联合应用后24,48和72h使MKN-45细胞凋亡率高于对照组(24h:5.73%,5.41%vs4.38%,P<0.01;48h:6.88%,6.32%vs4.35%,P<0.01;72h:7.72%,8.62%vs4.54%,P<0.01),低于缬草波春组(24h:5.73%,5.41%vs8.14%,P<0.01;48h:6.88%,6.32%vs12.31%,P<0.01;72h:7.72%,8.62%vs26.41%,P<0.01),与对照组及缬草波春组比较差异均有显著意义(P<0.01).Caspase-8抑制剂与缬草波春联合应用后24,48和72hMKN-45细胞凋亡率明显增加,与对照组比较差异有显著意义(8.02%vs4.38%,P<0.01;11.05%vs4.35%,P<0.01;24.86%vs4.54%,P<0.01),与单用缬草波春组比较差异无显著意义.缬草波春降低MKN-45胃癌细胞株SurvivinmRNA的表达,并有浓度依赖性和时间依赖性,而且使MKN-45胃癌细胞株P53蛋白表达增加,Survivin蛋白表达降低,均有浓度依赖性.结论:缬草波春可诱导MKN-45细胞凋亡,其作用可部分被Caspase-3,Caspase-9抑制剂所抑制,但不能被Caspase-8抑制剂所抑制.缬草波春诱导MKN-45胃癌细胞株凋亡与P53蛋白表达提高及SurvivinmRNA和Survivin蛋白低表达降低有关.
AIM： To study the apoptosis of gastric cancer cell line MKN-45 induced by valepotriate and its relationship with the expression of Caspase, P53, and Survivin.
METHODS： Gastric cancer cell line MKN-45 was divided into 4 groups, named group A （control）, B （treated with Caspase-3, -8 and -9 inhibitors）, C （treated with valepotriate） and D （treated with inhibitory agents plus valepotriate）, respectively. The apoptosis rates of MKN-45 cells were tested by fluorescence activated cell sorter （FACS） at different time （24, 48 and 72 h） in each group. After exposure to different concentrations of valepotriate for different time （12, 24, 48 and 72 h）, MKN-45 cells were collected and the RNA was extracted by tripure agent. The mRNA expression of Survivin was assayed by reverse transcription-polymerase chain reaction （RT- PCR）, while the protein expression of P53 and Survivin were detected by immunohistochemical methods 24 hours after exposure to different concentrations of valepotriate （50 and 100 mg/L）.
RESULTS： The apoptosis rates of MKN-45 cells were not significantly different between group A and B at 24, 48 and 72 h （P 〉 0.05）. The apoptosis rates were significantly higher in MKN-45 cells exposed to valepotriate plus Caspase-3 inhibitor or Caspase-9 inhibitor for 24, 48 and 72 h than those in group A （24 h： 5.73%, 5.41% vs 4.38%, P 〈 0.01; 48 h： 6.88%, 6.32% vs 4.35%, P 〈 0.01; 72 h： 7.72%, 8.62% vs 4.54%, P 〈 0.01）, but lower than those in group C （24 h： 5.73%, 5.41% vs 8.14%, P 〈 0.01; 48 h： 6.88%, 6.32% vs 12.31%, P 〈 0.01; 72 h： 7.72%, 8.62% vs 26.41%, P 〈 0.01）. The apoptosis rates of MKN-45 cells exposed to valepotriate plus Caspase-8 inhibitor for 24, 48 and 72 h were notably increased in comparison with those in group A （8.02% vs 4.38%, P 〈 0.01; 11.05% vs 4.35%, P 〈 0.01; 24.86% vs 4.54%, P 〈 0.01）, but was not significantly different from those in group C （P 〉 0.05）. Valepotriate down-regulated the expression of
出处
《世界华人消化杂志》
CAS
北大核心
2007年第1期22-28,共7页
World Chinese Journal of Digestology
