摘要
目的Survivin是近年来发现的凋亡抑制蛋白家族新成员,在多数恶性肿瘤组织中丰富表达。因此,观察Survivin反义寡核苷酸(ASODN)转染对人膀胱移行细胞癌T24细胞凋亡、增殖、细胞对化疗药物敏感性的影响。方法设计合成针对SurvivnmRNA特异性的ASODN,透射电镜观察细胞形态变化,流式细胞术检测各组细胞增殖指数(proliferativeindex,PI)和凋亡指数(apoptoticindex,AI),RT-PCR法分析转染后Sur-vivnmRNA及Caspase-3mRNA的变化,WesternBlot法检测各组Survivn蛋白表达情况,荧光分光光度法测定Caspase-3的活性水平;MTT实验测定T24细胞对丝裂霉素(mitomycin,MMC)的敏感性。结果A-SODN转染组透射电镜下观察到凋亡的征象,而各对照组未见凋亡证据;各转染组细胞AI明显高于各对照组,PI明显低于各对照组(P<0.01),且在一定范围内呈量-效关系,各对照组间差异无显著性(P>0.05);各转染组细胞SurvivinmRNA、Survivin的表达较各对照组明显下调(P<0.01),各对照组间差异无显著性(P>0.05);Caspase-3mRNA的表达在各组细胞间差异无显著性(P>0.05);各转染组细胞Caspase-3活化水平较各对照组明显提高(P<0.01),各对照组间差异无显著性(P>0.05);MTT实验显示,转染组MMC半数抑制浓度(IC50)较对照组明显下降,ASODN可提高T24细胞对MMC的敏感性。结论SurvivinASODN转染T24细胞后可特异性封闭Survivin的表达,对Caspase-3mRNA的表达无明显作用,能激活Caspase-3无活性前体,从而诱导T24细胞凋亡,抑制细胞增殖,提高T24细胞对化疗药物MMC的敏感性。
[Objective] Survivin is a recently described inhibitor of apoptsisis, It is undetectable in normal tissue of adults, but abundantly expressed in transformed cells and a variety of human cancers, This study was aimed to investigate the effect of Survivin antisense oligodeoxynucleotide （ASODN） targeting Survivin mRNA on human bladder transitional cell carcinoma（BTCC） T24 ceil growth, apoptosis, apoptosis related molecules, proliferation and sensitivity to chemotheraputic drug-mitomycin （MMC）. [Methods] ASODN targeting Survivn mRNA was designed and synthesized. Cell morphological change was observed with transmission electron microscope, apeptotic index （AI） and proliferative index （PI） were examined by flow cytometry, Survivn mRNA and Caspase-3 mRNA expression was detected by semi-quantitative polymerase chain reaction （RT-PCR）, Survivn protein expression was detected by Western Blot, the level of activity of Caspase-3 was measured by spectrophotography; MTF assay was performed to evaluate the sensibihty of T24 cells to MMC. [Results] Abnormal cell morphological change was observed in the ASODN transfected group, no apoptotic evidences were observed in the control groups. The AI of ASODN group was significantly higher than that of other groups, the PI of ASODN group was significantly lower than that of other groups, there was does-effect relation in some coverage, no significant difference was observed among the control groups. Expression of Survivin mRNA and Survivin protein in ASODN group was significantly decreased compared with that in control groups, expression of Caspase-3 mRNA showed no significant difference among the groups. The level of activity of Caspase-3 in ASODN group was significantly increased than that of other groups. Compared to control groups, the half inhibition concentration （IC50） of MMC to T24 cells in ASODN group was significantly reduced by MTT assay, the difference was significant by statistical analysis. [Conclusion] Survivin ASODN transfection may decrease t
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2006年第13期1955-1960,共6页
China Journal of Modern Medicine
