摘要
目的观察caspase-3 mRNA反义寡核苷酸(ASODN)对HL-60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-3 mRNA不同序列的4条ASODN导入HL-60细胞中,γ-射线照射。应用电泳法检测DNA梯状条带;Hoechst 33258-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-3 mRNA5’非编码区(-62至-46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<0.01),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<0.05)。结论caspase-3 mRNA ASODN可抑制γ-射线诱导的HL-60细胞凋亡,作用有序列特异性及剂量依赖性。该结果为细胞过度凋亡相关疾病的基因治疗提供了实验基础。
Objective To observe the inhibitory effect of caspase-3 rnRNA antisense oligodeoxynucleotides （ASODNs） on the apoptosis of HL-60 cells, and to screen the effective ASODN. Methods By means of lipofectimine-mediating transfected, caspase-3 mRNA ASODNs of four different sequences were introduced into HL-60 cells followed by y-irradiation. The gel electrophoresis was performed for the DNA ladder, Hoechst 33258-propidium iodide fluorescent staining for the percentage of apoptotic cells, and flow cytometry （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 ≥3μmol/L, the DNA ladder was not detected with 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 the nontransfection group and the mismatched oligodeoxynuleotide group （P〈0. 01）. With the increase in 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 ASODN （P〈0. 05）. Conclusion Caspase-3 mRNA ASODN can inhibit y-irradiation-induced apoptosis of HL-60 cells, the effect being sequence-specific and dose-dependent. The findings may lay an experimental foundation for gene therapies of diseases related to excessive apoptosis.
出处
《中国组织化学与细胞化学杂志》
CAS
CSCD
2006年第1期1-6,共6页
Chinese Journal of Histochemistry and Cytochemistry
基金
ThisstudyissupportedbytheNationalNaturalScienceFoundationofChina(39880008)
