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为消除白血病细胞中ｂｃｌ－２基因对细胞凋亡的抑制作用，开辟白血病基因治疗的途径。将合成的针对ｂｃｌ－２ｍＲＮＡ的“锤头型”核酶（Ｒｉｂｏｚｙｍｅ，ＲＺ）基因定向克隆于真核表达载体ｐＤＯＲ－ｎｅｏ，构成ｐＤＯＲ－ＲＺ重组体。通过脂质体Ｌｉｐｏｆｅｃｔｉｎ介导的ＤＮＡ转染法，把ｐＤＯＲ－ＲＺ导入ＨＬ－６０细胞。用Ｓｏｕｔｈ－ｅｒｎ印迹，ＲＮＡ斑点杂交法观察ＲＺ基因在ＨＬ－６０细胞内表达，并通过电镜、流式细胞仪（ＦＣＭ），光镜和ＤＮＡ电泳观察ＲＺ对ＨＬ－６０细胞的影响。结果：①ＲＺ在转染ＨＬ－６０后７２小时得以表达，细胞内ｂｃｌ－２蛋白合成受到抑制。②在ＦＣＭ图谱上可见到明显的凋亡峰，形态观察ＲＺ处理的ＨＬ－６０细胞呈典型的凋亡形态。③足叶乙甙（促凋亡）敏感性试验表明，与未转染的细胞和转染空载体ｐＤＯＲ－ｎｅｏ的细胞相比，转染ｐＤＯＲ－ＲＺ的细胞ＤＮＡ电泳易出现梯状条带。结果说明ＲＺ基因通过逆转录病毒表达载体导入ＨＬ－６０细胞后可成功地表达并抑制ＨＬ－６０细胞ｂｃｌ－２蛋白的合成，并促进ＨＬ－６０细胞凋亡
To eliminate the inhibitory effect of bcl-2 gene on apoptosis in leukemia cells and to open up the way of gene therapy for leukemia. The synthesized Ribozyme (RZ) gene targeting bcl-2mRNA was cloned into the eukaryotic expression vector pDOR-neo to construct the recombinant pDOR-RZ. PDOR-RZ was introduced into HL-60 cells by Lipofectin-mediated DNA transfection. The expression of RZ gene in HL-60 cells was observed by South-ern blotting and RNA dot blot hybridization. The effects of RZ on HL-60 cells were observed by electron microscopy, flow cytometry (FCM), light microscopy and DNA electrophoresis. Results: ①RZ was expressed 72h after transfection with HL-60, and the bcl-2 protein synthesis was inhibited. ② The obvious apoptotic peak was seen on the FCM. HL-60 cells treated with RZ showed a typical apoptotic morphology. ③ Etoposide (apoptosis) sensitivity test showed that, compared with untransfected cells and cells transfected with empty vector pDOR-neo, DNA ladder electrophoresis appeared in the cells transfected with pDOR-RZ. The results showed that RZ gene could be successfully expressed in HL-60 cells by retroviral expression vector and inhibited the synthesis of bcl-2 protein in HL-60 cells and promoted the apoptosis of HL-60 cells