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目的选用２０ｎｔＢｃｌ－２反义硫代磷酸寡核苷酸（Ａｓ－Ｐｓ－ＯＤＮ，ＡＳＰＯ），并以其正义寡核苷酸（ＳＰＯ）为对照做如下问题探讨：（１）普通剂量（５～２０μｍｏｌ）ＡＳＰＯ对ＨＬ６０细胞、正常及缺铁性贫血患者骨髓细胞及急性白血病病人骨髓细胞或外周白血病细胞（幼稚细胞≥８５％）的作用；（２）大剂量（１６０μｍｏｌ）正义或反义寡核苷酸对ＨＬ６０细胞作用的特点，并通过ＡＳＰＯ引起ＨＬ６０细胞Ｂｃｌ－２蛋白表达变化和ＳＰＯ对ＨＬ６０细胞毒性的变化，寻找可能的最佳效应剂量和最小毒性剂量；（３）ＡＳＰＯ联合化疗药物对ＨＬ６０细胞和原代急性白血病细胞的作用。方法台盼蓝拒染试验和克隆培养测定细胞的生长能力；免疫细胞化学染色和流式细胞仪检测Ｐ２６Ｂｃｌ－２蛋白表达，ＲＴ－ＰＣＲ法检测Ｂｃｌ－２ｍＲＮＡ（Ｂｃｌ－２／β－ａｃｔｉｎ）相对水平；光、电镜下观察凋亡细胞形态，吖啶橙染色及流式细胞仪检测细胞凋亡率，ＤＮＡ提取及电泳观察凋亡细胞ＤＮＡ降解片段的形成；ＭＴＴ法检测ＰＳ－ＯＤＮ及化疗药物的细胞毒作用。结果（１）普通剂量的ＡＳＰＯ即能降低ＨＬ６０细胞及白血病细胞Ｂｃｌ－２ｍＲＮＡ的表达，并抑制ＨＬ６０细胞及１８／２０例白血病细?
Objective To select 20 nt Bcl-2 antisense phosphorothioate oligodeoxynucleotide (As-Ps-ODN, ASPO) and use its sense oligonucleotide (SPO) as a control for the following questions: (1) Ordinary dose (5- 20μmol) of the effect of ASPO on HL60 cells, bone marrow cells in patients with normal and iron-deficiency anemia, and bone marrow cells or peripheral leukemia cells (≥ 85% of immature cells) in patients with acute leukemia; (2) large dose (160μmol) of sense or antisense oligonucleotides The characteristics of glucuronide on HL60 cells, and through ASPO induced changes in Bcl-2 protein expression in HL60 cells and changes in the toxicity of SPO to HL60 cells, looking for possible optimal effect dose and minimal toxic dose; (3) ASPO combined with chemotherapy drugs The role of HL60 cells and primary acute leukemia cells. Methods Trypan blue exclusion assay and clone culture were used to determine the growth ability of the cells; P26Bcl-2 protein expression was detected by immunocytochemistry and flow cytometry; and Bcl-2 mRNA (Bcl-2/β-actin) was detected by RT-PCR. The level of apoptotic cells was observed under light and electron microscope. Apoptotic rate of cells was detected by acridine orange staining and flow cytometry. DNA was extracted and electrophoresed to observe the formation of apoptotic DNA degradation fragments. PS-ODN and chemotherapeutic drugs were detected by MTT assay. The cytotoxic effect. Results (1) The normal dose of ASPO can reduce the expression of Bcl-2 mRNA in HL60 cells and leukemia cells, and inhibit the HL60 cells and 18/20 cases of leukemia.