摘要
目的 观察caspase 3mRNA反义寡核苷酸 (ASODN)对HL 6 0细胞凋亡的抑制作用 ,筛选有效ASODN。 方法 用脂质体介导法将针对caspase 3mRNA不同序列的 4条ASODN导入HL 6 0细胞中 ,γ 射线照射。应用电泳法检测DNA梯状条带 ;Hoechst332 5 8 碘化丙啶染色 ,荧光显微镜分析凋亡细胞百分率 ;流式细胞术进行细胞凋亡定量。 结果 以caspase 3mRNA 5′非编码区 (- 6 2至 - 4 6位 )与编码起始区 (- 1至 16位 )ASODN转染 ,当转染终浓度≥ 3μmol L时 ,DNA电泳梯状条带消失 ,流式细胞术亦未见明显的亚二倍体峰 ;荧光染色分析 ,凋亡细胞百分率比未转染对照组和错配寡核苷酸对照组显著降低 (P <0 0 1) ,且随转染终浓度的增加 ,凋亡抑制率显著增加。另外 ,5′非编码区ASODN的抑制作用显著强于编码起始区ASODN(P <0 0 5 )。 结论 caspase 3mRNAASODN可抑制γ 射线诱导的HL 6 0细胞凋亡 。
Objective To observe the inhibitory effect of caspase\|3 mRNA antisense oligodeoxynucleotides (ASODN) on the apoptosis of HL\|60 cells, and to screen the effective ASOND. Methods By means of the lipofectimine\|mediating transfection, caspase\|3 mRNA ASODNs of four different sequences were introduced into HL\|60 cells followed by γ irradiation.The gel electrophoresis was performed for the DNA ladder, Hoechst 33258\|propidium iodide fluorecent staining for the percentage of apoptotic cells, and FCM for quantitative analysis of apoptosis. Results When HL\|60 cells were transfected with caspase\|3 mRNA ASODNs targeting 5'noncoding region (sites -62 to -46) and initiative coding region (sites -1 to 16) at the final concentration of ≥3?μ mol/L, the DNA ladder was not detected with the gel electrophoresis, nor was the apoptotic peak found with FCM. Fluorescent staining analysis showed that the percentage of apoptotic cells was significantly lower than that in the control groups including non\|transfection group and mismatched oligodeoxynuleotide group ( P <0 01).With increase of the final transfection concentration, the inhibition rate of apoptosis was increased markedly. Furthermore, the noncoding region ASODN was more effective than the initiative coding region ASOND ( P <0 05).Conclusion Caspase\|3 mRNA ASODN can inhibit γ irradiation\|induced apoptosis of HL\|60 cells, the effect being sequence specific and dose dependent. [
出处
《解剖学报》
CAS
CSCD
北大核心
2003年第2期172-176,共5页
Acta Anatomica Sinica
基金
国家自然科学基金资助项目 ( 3 9880 0 0 8)
陕西省自然科学基金资助项目 ( 98sm61)
