【作者】 张凯；

【导师】 魏素菊；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2009， 硕士

【摘要】 目的:采用体外实验,研究磷酸肌酸钠(Creatine Phosphate Sodium,CP)对人卵巢癌细胞株skov3侵袭、迁移所产生的作用。观察磷酸肌酸钠对skov3细胞株侵袭、迁移影响的结果,探讨磷酸肌酸钠影响肿瘤细胞侵袭、迁移的可能机制,为应用该药物治疗化疗心脏毒性提供实验依据。方法:体外培养人卵巢癌细胞株skov3,分为空白对照组及不同浓度的药物处理组。取生长状态良好的对数期细胞,采用四甲基偶氮唑蓝比色法(MTT比色法)检测磷酸肌酸钠对人卵巢癌细胞株skov3黏附能力的影响;采用Transwell小室法检测磷酸肌酸钠对该细胞株迁移能力、侵袭能力的影响作用;采用流式细胞仪测定磷酸肌酸钠对该细胞株nm23-h1、c-myc、MMP-2、MMP-9蛋白表达水平的影响;采用逆转录-聚合酶链反应(revers transcription PCR,RT-PCR)半定量检测磷酸肌酸钠对该细胞株nm23-h1、c-myc mRNA表达水平的影响。结果:MTT比色法分析检测黏附能力实验显示:skov3细胞株药物处理组(1、6、12mmol/L)与空白对照组相比,低浓度药物处理组OD值无显著变化,结果无统计学差异(P>0.05),中、高浓度药物处理组OD值均出现显著下降,结果有统计学差异(P<0.05),但中、高浓度药物处理组之间OD值无显著变化,结果无统计学差异(P>0.05);计算粘附抑制率分别为0%,0.72%±0.92%,36.47%±3.17%,37.06%±3.30%,低浓度药物处理组的细胞粘附抑制率较空白组无显著变化,结果无统计学差异(P>0.05),中、高浓度药物处理组的细胞粘附抑制率较空白组显著升高,结果有统计学差异(P<0.05),但组间无显著变化,结果无统计学差异(P>0.05);即中、高浓度药物处理组对skov3细胞株与细胞外基质成分Matrigel的粘附作用均有显著抑制。侵袭实验显示:skov3细胞株药物处理组(1、6、12mmol/L)与空白对照组比较,低浓度药物处理组穿膜细胞数较空白对照组无显著变化,结果无统计学差异(P>0.05),中、高浓度药物处理组穿膜细胞数量较空白对照组均显著降低,结果有统计学差异(P<0.05),但中、高浓度药物处理组之间穿膜细胞数量无显著变化,结果无统计学差异(P>0.05);计算侵袭抑制率分别为0%、3.06%±4.84%、34.87%±3.19%、37.67%±6.06%;即中、高浓度药物处理组对skov3细胞株的侵袭能力有显著性抑制作用。迁移实验显示:skov3细胞株药物处理组(1、6、12mmol/L)与空白对照组比较,低浓度药物处理组穿膜细胞数较空白对照组无显著变化,结果无统计学差异(P>0.05),中、高浓度药物处理组穿膜细胞数量较空白对照组均显著降低,结果有统计学差异(P<0.05),但中、高浓度药物处理组之间穿膜细胞数量无显著变化,结果无统计学差异(P>0.05);计算迁移抑制率分别为0%、2.32%±1.47%、31.50%±2.23%、33.51 %±2.90%;即中、高浓度药物处理组对skov3细胞株的迁移能力有显著性抑制作用。流式细胞仪测定细胞蛋白表达:skov3细胞株药物处理组(1、6、12mmol/L)采用流式细胞仪测定结果,以荧光指数(FI)表示四种蛋白表达的相对含量。所有样品检测均以0mmol/L为空白对照组,计算各样品FI值。nm23-H1的FI值分别为1、1.00±0.07、1.27±0.07、1.28±0.05;c-myc的FI值分别为1、1.00±0.04、0.87±0.01、0.85±0.03;MMP-2的FI值分别为1、0.99±0.04、0.98±0.06、0.98±0.03;MMP-9的FI值分别为1、1.01±0.03、0.99±0.03、0.98±0.05;即中、高浓度药物处理组skov3细胞株nm23-h1蛋白的表达水平与空白对照组相比显著升高,结果有统计学差异(P<0.05);中、高浓度药物处理组skov3细胞株c-myc蛋白的表达水平与空白对照组相比显著降低,结果有统计学差异(P<0.05);但各浓度药物处理组skov3细胞株MMP-2、MMP-9蛋白的表达水平与空白对照组相比均无显著变化,结果无统计学差异(P>0.05)。半定量RT-PCR检测结果显示:skov3细胞株药物处理组(1、6、12mmol/L)采用半定量RT-PCR检测nm23-h1、c-myc mRNA的表达水平,药物处理组与空白对照组均可见明显而清晰的特异性条带。应用美国FOTODYNE凝胶成像分析系统分析显示,反映nm23-H1 mRNA表达水平的nm23-H1/GAPDH值分别为0.41±0.02、0.42±0.02、0.63±0.04、0.65±0.03 ,反映C-myc mRNA表达水平的C-myc/GAPDH值分别为0.63±0.02、0.62±0.03、0.40±0.01、0.39±0.03;即中、高浓度药物处理组skov3细胞株nm23-h1 mRNA的表达水平与空白对照组相比显著升高,结果有统计学差异(P<0.05);中、高浓度药物处理组skov3细胞株c-myc mRNA的表达水平与空白对照组相比显著降低,结果有统计学差异(P<0.05)。结论:1一定浓度的磷酸肌酸钠具有抑制人卵巢癌细胞株skov3黏附、迁移、侵袭的作用。2一定浓度的磷酸肌酸钠可通过上调nm23-h1 mRNA和蛋白表达水平抑制人卵巢癌细胞株skov3黏附、迁移、侵袭。3一定浓度的磷酸肌酸钠可通过下调c-myc mRNA和蛋白表达水平抑制人卵巢癌细胞株skov3黏附、迁移、侵袭。4磷酸肌酸钠对MMP-2、MMP-9蛋白表达水平无显著影响。5磷酸肌酸钠对卵巢癌细胞株skov3的侵袭、迁移无促进作用,为应用该药物治疗化疗心脏毒性提供了实验依据。更多还原

【Abstract】 Objective: To study the effects of Creatine Phosphate Sodium For Injection(CP)on invasion and migration of human ovarian cancer cell line SKOV3 and its mechanisms. Meanwhile, to investigate the possible mechanisms of CP inhibit invasion、adhesion and migration of SKOV3 cells; and Provide experiment evidence for use CP to treat the anthracycline-induced cardiotoxicity.Methods: SKOV3 cells were incubated in culture medium in vitro, divide into untreated group and treated group(1mmol/l、6mmol/L、12mmol/L). Effect of CP on the adhesion of SKOV3 cells was measured by MTT colorimetric method. Effect of CP on the invasion and migration of SKOV3 cells was measured by transwell method. The expression of nm23-h1、c-myc、MMP-2、MMP-9 protein in SKOV3 cells was analyzed by flow cytometric indirect immunofluorescent technigue. The expression of nm23-h1、c-myc gene in SKOV3 cells was analyzed by RT-PCR .Results: After treated with CP(1、6、12mmol/L )for 60min, measure the ability of adhesion by MTT colorimetric method. The adhesion rate was 0% in untreated group. The adhesion rate was 0.72%±0.92% in CP-treated group (1mmol/l). The adhesion rate was no significant higher, there was no statistically significant difference (P>0.05). The adhesion rate were 36.47%±3.17% in CP-treated group (6mmol/l) and 37.06%±3.30% in CP-treated group (12mmol/l). These two adhesion rates were significant higher than the untreated group, there were statistically significant difference (P<0.05), but there was no statistically significant difference in these two adhesion rates(P>0.05). The results show that CP inhibited adhesion of SKOV3 cells in the range of 6-12mmol/L.After treated with CP(1、6、12mmol/L), test the ability of migration in SKOV3 cells. In migratory experiments, compare with 0mmol/L concentration, the invaded cell numbers in 1mmol/L was no significant lower, there was no statistically significant difference(P>0.05), those numbers in 6 mmol/L and 12mmol/L concentration were statistically significant difference (P<0.05),.but there were no statistically significant difference between 6 mmol/L and 12mmol/L concentration(P>0.05). The Inhibition rates were 0% in untreated group, 3.06%±4.84% in CP-treated group(1mmol/l), 34.87%±3.19% in CP-treated group(6mmol/l), 37.67%±6.06% in CP-treated group (12mmol/l).After treated with CP(1、6、12mmol/L),test the ability of invasion in SKOV3 cells. In invasive experiments,compare with 0mmol/L concentration,the invaded cell numbers in 1mmol/L was no significant lower, there was no statistically significant difference(P>0.05), those numbers in 6 mmol/L and 12mmol/L concentration were statistically significant difference (P<0.05),but there were no statistically significant difference between 6 mmol/L and 12mmol/L concentration(P>0.05).The Inhibition rates were 0% in untreated group, 2.32%±1.47% in CP-treated group(1mmol/l), 31.50%±2.23% in CP-treated group(6mmol/l), 33.51%±2.90% in CP-treated group (12mmol/l).FCM assay results:The expression of nm23-H1、c-myc、MMP-2、MMP-9 protein had been examined in SKOV3 cells being treated with 0、1、6、12mmol/L CP for 72h, the FI-value of nm23-H1 in SKOV3 was 1、1.00±0.07、1.27±0.07、1.28±0.05;the FI-value of c-myc in SKOV3 was 1、1.00±0.04、0.87±0.01、0.85±0.03. To the FI values of nm23-H1 protein, there was statistically significant difference between control group and CP(6、12 mmol/L) group(P<0.05),but there was no statistically significant difference between control group and CP(1 mmol/L) group(P>0.05). To the FI values of c-myc protein, there was statistically significant difference between control group and CP(6、12 mmol/L) group(P<0.05),but there was no statistically significant difference between control group and CP(1 mmol/L) group(P>0.05).But the FI values of MMP-2、MMP-9 protein, there were no statistically significant difference between control group and CPgroup(P>0.05).RT-PCR detection results:After treated with CP(1、6、12mmol/L) for 72h,compared with control group, the expression of c-myc mRNA decreased, and there was statistically significant difference between control group and CP(6、12 mmol/L) group(P<0.05), but there was no statistically significant difference between control group and CP(1 mmol/L) group ( P > 0.05 ) .And compared with control group,the expression of nm23-H1 mRNA increased, and there was statistically significant difference between control group and CP(6、12 mmol/L) group(P<0.05), but there was no statistically significant difference between control group and CP(1 mmol/L) group(P>0.05).Conclusions: 1 CP inhibited the ability of adhesion、invasion and migration in SKOV3 cells.2 CP can increase the expression of nm23-H1 in SKOV3 cells. We infer that CP’s effects of inhibiting adhesion、invasion and migration in SKOV3 cells seem to due to up-regulation of the expression of nm23-h1 mRNA and protein.3 CP can reduce the expression of C-myc in SKOV3 cells. We infer that CP’s effects of inhibiting adhesion、invasion and migration in SKOV3 cells seem to due to down-regulation of the expression of C-myc mRNA and protein. 4 CP hasn’t effect the expression of MMP-2、MMP-9 in SKOV3 cells.5 CP can’t promote the adhesion、invasion and migration in SKOV3 cells in vitro,Provide experiment evidence for use CP to treat the anthracycline-induced cardiotoxicity.更多还原