Apollon siRNA联合川芎嗪对白血病细胞增殖及凋亡的影响

Effect of Apollon siRNA combined with tetramethylpyrazine on prolife-ration and apoptosis of leukemia K562 cells

摘要:

目的 观察Apollon siRNA 靶向沉默Apollon 基因联合川芎嗪（TMP）对人慢性粒细胞白血病细胞株K562 增殖及凋亡的影响。方法 根据前期实验筛选出的干扰效果最佳的Apollon siRNA 片段，构建pGPHIGFP-Neo-Apollon 真核表达载体，并将构建成功的载体转染至K562 细胞。将实验分为细胞对照组（未行任何处理）、阴性对照组（转染阴性质粒载体）和RNA 干扰组（转染pGPHI-GFP-Neo-Apollon 质粒载体），利用RT-PCR 法和细胞免疫荧光法分别检测各组细胞Apollon mRNA 及蛋白的表达情况；再于上述分组基础上新增TMP 组（施加320 μg/mL TMP）、TMP+ 阴性对照组和TMP+RNA 干扰组，应用MTT 法和流式细胞术分别检测各组K562 细胞的增殖能力和细胞凋亡率。结果 构建的pGPHI-GFP-Neo-Apollon 载体能在K562 细胞内稳定表达；RNA 干扰组Apollon mRNA 及其蛋白的相对表达水平明显低于细胞对照组和阴性对照组（均P<0.05）；RNA 干扰组K562细胞的增殖抑制率和凋亡率高于细胞对照组（P<0.05），与RNA 干扰组及TMP 组比较，siRNA 转染联合TMP能显著提高K562 细胞的增殖抑制率和凋亡率（均P<0.05）。结论 Apollon siRNA 转染能显著抑制K562 细胞增殖并促进其凋亡，且与TMP 联合使用对提高K562 细胞的增殖抑制率和凋亡率具有显著协同作用，提示siRNA技术联合药物在白血病治疗中具有重要的潜在价值。

Abstract:

Objective To investigate the effect of small interfering RNA (siRNA) silencing Apollon gene combined with tetramethylpyrazine (TMP) on the proliferation and apoptosis of human chronic myeloid leukemia cell line K562. Methods K562 cells were divided into blank control, negative control, and RNA interference (RNAi) group. For the RNAi group, the pGPHI-GFP-Neo-Apollon eukaryotic expression vector based on the best Apollon siRNA fragments screened out in previous experiments was constructed; the blank control group received no treatment, and the negative control group was transfected with negative plasmid vector. The mRNA and protein expression of Apollon was measured by RT-PCR and cell immunofluorescence, respectively. Additionally, TMP (320 μg/mL) was applied to set TMP, TMP+negative control, and TMP+RNAi groups. The cell viability and apoptosis rate were determined by MTT assay and flow cytometry, respectively. Results The constructed vector was stably expressed in K562 cells. The RNAi group had significantly lower mRNA and protein expression of Apollon than the blank control group and negative control (P<0.05). The RNAi group had significantly increased proliferation inhibition rate and apoptosis rate, as compared with the blank contorl group (P<0.05). The TMP+RNAi group had significantly increased proliferation inhibition rate and apoptosis rate, as compared with the RNAi, and TMP groups (P<0.05). Conclusions Apollon siRNA can significantly inhibit the proliferation and promote the apoptosis of K562 cells, and the addition of TMP can further increase the proliferation inhibition rate and apoptosis rate, suggesting that siRNA technology combined with drugs has a significant potential value in the treatment of leukemia.