Bcl-2反义核酸联合化疗药对HL60细胞及原代白血病细胞的作用

目的应用Bcl-2反义硫代磷酸寡脱氧核苷酸(anti-sense phosphorothioate oligodeoxynucleotide,AS-PS-ODNs,AS-PO)靶向诱导20例原代急性白血病细胞凋亡,并与多种常用化疗药联合,观察ASPO对原代急性白血病细胞及HL60细胞系的协同效应。方法①应用MTT比色法分析ASPO与常用化疗药物配伍协同作用特点;②细胞免疫化学染色检测P26 Bcl-2蛋白表达;③台盼蓝拒染试验检测白血病细胞倍增时间;④丫啶橙(AO)染色,流式DNA Content分析细胞凋亡率,电镜及DNA电泳观察凋亡细胞形态及分子水平的变化。结果①ASPO与除DNR,NVT外的7种(HHRT,VCR、MTX、Ara-c、VP16、ACR、DEX)化疗药物均有联合增强或相加作用。②12例Bcl-2蛋白抑制率大于0.4为A组,8例小于0.4为B组。A组的凋亡率和B组的凋亡率均高于空白对照组(P<0.05),且A组的凋亡率高于B组(P<0.05)。③原代急性白血病细胞ASPO组,Ara-c组和ASPO+Ara-c组,72 h细胞生长抑制检测发现ASPO+Ara-c组的细胞抑制率高于单用ASPO与单用Ara-c的细胞抑制率之和(P<0.01);④A组的ASPO对细胞的抑制与细胞生长的倍增时间102.89±37.97呈正相关γ=0.577(P<0.05)。结论Bcl-2 ASPO能特异抑制白血病细胞(系)的Bcl-2蛋白表达;Bcl-2 ASPO对倍增时间慢、BCL-2蛋白表达高的原代白血病细胞作用效果好;Bcl-2的ASPO与多种化疗药物联合有潜在临床应用前景。

The effect of the target of Bcl-2 antisense phosphorothioate oligodexynucleotides combined with chemotherapy on HL 60 cell line and the primary leukemia cells

Aim To investigate the effect of the target Bcl-2 antisense phosphorothioate oligodeoxynucleotide(AS-PS-ODNs,ASPO) inducing apoptosis and combining with the drugs of chemotherapy on HL60 cell line and 20 cases of primary acute leukemia(AL) cells.Methods The cellular toxicity of HL60 cell line resulted from application of the ASPO or combination with chemotherapeutic drugs was detected by MTT.The expression of Bcl-2 protein was determined by immunocytochemistry.The cell viability and proliferation were tested by Trypan Blue exclusion.The morphologic signs and the DNA ladder of apoptotic cells were observed by acridine orange staining,flow cytometry,transmission electron microscope and DNA fragment electrophoresis.Results ① The ASPO could work in coordination with many chemotherapeutic drugs,including HHRT,VCR,MTX,Ara-c,VP16,ACR and DEX in HL60 cell line.② In presence of the common doses(5 μmol·L-1) of ASPO,12 of 20 of primary ALL in which the suppression ratio of expression of Bcl-2 protein was more than 0.4 were set as group A,and 8 of 20 ones in which the suppression ratio of expression of Bcl-2 protein was lower than 0.4 were set as group B.The apoptotic ratio of group A was higher than that of group B(P<0.05),and the apoptotic ratio of both group A and group B was higher than that of control(P<0.05).③ The proliferation ratio of primary AL cells in presence of ASPO+Ara-c was suppressed than that of only presence of ASPO,or Ara-c,or plus of them(P<0.01).④ The suppression ratio of primary AL cells in presence of ASPO in group A was positively correlated to the redouble time(102.89±37.97) of the growth of primary AL cells(γ=0.577,P<0.05).Conclusions Bcl-2 ASPO is a sequence-specific inhibitor of Bcl-2 protein expression of primary AL cells and HL60 cell line.Bcl-2 ASPO is especially suitable for treating ALs which have lower proliferation and higher expression of Bcl-2 protein.The combination of Bcl-2 ASPO with chemotherapeutic drugs has a potential further application for treating the patients with acute leukemia.