hTERT基因反义核酸对顺铂诱导Jurkat细胞凋亡的影响

Effect of hTERT antisense oligodeoxynucleotide on Cis-diamminedichicloroplatinum-induced apoptosis in Jurkat cells

目的 :用人类端粒酶逆转录酶 (hTERT)基因的反义寡核苷酸 (ASODN)抑制Jurkat细胞端粒酶活性后 ,观察化疗药物 (顺铂、柔红霉素、长春新碱、足叶乙甙 )对Jurkat细胞凋亡的影响。方法 :采用台盼蓝拒染法观察hTERTASODN与化疗药物 (顺铂、柔红霉素、长春新碱、足叶乙甙 )联合作用对Jurkat细胞系生长的影响 ;姬姆萨染色法观察凋亡细胞的形态变化 ;琼脂糖凝胶电泳分析细胞凋亡的DNA断裂情况 ;通过流式细胞仪对细胞凋亡峰进行定量分析。结果 :hTERTASODN作用于Jurkat细胞 2 4h再加入柔红霉素、长春新碱、足叶乙甙 ,对细胞生长的抑制分别与单用柔红霉素、长春新碱、足叶乙甙及hTERT正义寡核苷酸 (senseoligodeoxynucleotide,SODN)联合柔红霉素、长春新碱、足叶乙甙组相比 ,无显著差异 (P >0 0 5 )。hTERTASODN作用于Jurkat细胞 2 4h再加入顺铂 ,分别与SODN联合顺铂作用组、单用顺铂作用组相比 ,对细胞抑制明显增强 (P <0 0 5 )。hTERTASODN作用于Jurkat细胞 2 4h再加入顺铂作用 4 8h ,细胞出现典型的凋亡形态学改变 ,经琼脂糖凝胶电泳即可见到DNA梯形条带 ,而SODN与顺铂联合作用组及单独应用顺铂均未见到DNA梯形条带。hTERTASODN与 2 5 μmol/L顺铂联合作用于Jurkat细胞 4 8h的凋亡细胞百分率 (2 5 18±

AIM: To explore the effect of hTERT antisense phosphorothioate oligodeoxynucleotide (ASODN) on apoptosis induced by chemotherapeutic drugs in Jurkat cell lines. METHODS: Cell viability was determined using the trypan blue dye exclusion assay. Apoptosis was detected by morphological observation, DNA gel electrophoresis and flow cytometry analysis. RESULTS: The survival rates of Jurkat cells cultured with daunorubicin, vincristin, and etoposide, respectively were similar with that cultured with those chemotherapic drugs plus hTERT ASODN. The survival rates of Jurkat cells cultured with cis-diamminedichicloroplatinum(DDP) added 24 hours later were higher than that cultured with hTERT ASODN and DDP added 24 hours later. The survival rates of Jurkat cells cultured with DDP were similar with that cultured with hTERT SODN and DDP. In morphological observation of apoptotic cells using Giemsa staining, cells displayed classic apoptotic changes treated with DDP or DDP combined with hTERT ASODN or SODN at 48 hours. Agarose gel electrophoresis of genomic DNA from Jurkat cells treated with ASODN and DDP combination for 48 hours showed typical DNA “ladder”. Neither the DNA from Jurkat cells treated with SODN plus DDP nor the DNA from the cells treated with DDP alone showed ladder pattern. Apoptosis rates of Jurkat cells treated with DDP for 48 hours after 24 hours of exposure to ASODN significantly increased. There was significant difference in the percentage of apoptotic Jurkat cells between hTERT ASODN plus DDP and SODN plus DDP or DDP alone, respectively. CONCLUSION: The hTERT ASODN complementary to the translation initiation region of hTERT mRNA enhanced DDP-induced apoptosis in Jurkat cells.