RNA干扰抑制Apollon表达联合川芎嗪可增强白血病细胞对化疗药物的敏感性

The expression of Apollon gene silenced by RNA interference combined with ligustrazine intervention may increase the sensitivity of leukemia cells to chemotherapeutic agents

目的:探讨RNA干扰(RNA interference,RNAi)技术沉默凋亡抑制蛋白Apollon基因表达联合中药川芎嗪(tetramethylpyrazine,TMP)能否增强人髓系白血病K562细胞对长春新碱(vincristine,VCR)和柔红霉素(daunorubicin,DNR)的药物敏感性。方法:依据前期实验筛选出的干扰效果最佳的靶向Apollon的小干扰RNA(small interference RNA,siRNA)片段,构建pGPHI-GFP-Neo-Apollon真核表达载体,应用LipofectAMINETM2000转染白血病K562细胞,G418稳定筛选。采用反转录-PCR(reverse transcriptase PCR,RT-PCR)法和细胞免疫荧光法分别检测重组载体稳定转染K562细胞后Apollon mRNA及其蛋白的表达情况;RNAi联合TMP与VCR或DNR用药后,应用MTT法检测细胞对化疗药物敏感性的变化,FCM法检测细胞凋亡率。结果:成功构建了pGPHI-GFP-Neo-Apollon载体并在K562细胞内稳定表达,重组载体能有效沉默Apollon mRNA及蛋白表达。RNAi组细胞联合TMP作用后能明显增加细胞对VCR、DNR的敏感性,其半数抑制浓度(half-inhibitory concentration,IC50)值明显低于单纯TMP作用组(P<0.05),细胞凋亡率也显著增加,与未经任何处理的对照组及单纯TMP作用组比较的凋亡率差异有统计学意义(P<0.05)。结论:靶向Apollon的RNAi联合TMP能明显提高K562细胞对VCR、DNR的敏感性,显著减少化疗药物使用剂量且更易于化疗药物诱导细胞凋亡,提示RNAi沉默Apollon基因联合中药TMP能在一定程度上逆转白血病细胞的耐药性。

Objective： To investigate whether the expression of Apollon （an apoptosis inhibitory gene） silenced by using RNA interference （RNAi） technology in combination with tetramethylpyrazine （TMP） intervention can increase the sensitivity of human myeloid leukemia K562 cells to vincristine （VCR） and daunorubicin （DNR）. Methods： Using the best small interference RNA （siRNA） targeting Apollon gene which was screened out in a previous experiment, the eukaryotic expression vector pGPHI-GFP-Neo-Apollon was constructed. Then, the K562 cells were transfected with pGPHI-GFP-Neo-Apollon by LipofectamineTM 2000 and screened out by G418. After Apollon siRNA was transfected stably into K562 cells, the expressions of Apollon mRNA and protein were detected by reverse transcription （RT）-PCR and immunofluorescence, respectively. The sensitivities to chemotherapeutic agents VCR and DNR after RNAi in combination with TMP intervention were detected by MTT method, and apoptosis rate was detected by flow cytometry. Results： pGPHI-GFP-Neo-Apollon vector was constructed successfully and it could express stably in K562 cells, which silenced the expressions of Apollon mRNA and protein effectively. The sensitivity to VCR or DNR increased significantly after RNAi in combination with TMP intervention. The half inhibitory concentration （IC50） values of VCR and DNR were significantly lower than those in the group of TMP intervention alone （P 〈 0.05）. The apoptosis rate of K562 cells after transfection with Apollon siRNA in combination with TMP intervention was significantly increased comparing with that of the untreated control group and TMP intervention alone group （P 〈 0.05）. Conclusion： RNAi targeting Apollon gene in combination with TMP intervention can obviously increase the sensitivity to VCR or DNR in K562 cells and significantly reduce the dosage of chemotherapeutic agents as well as improve apoptosis induced by chemotherapy. These results suggest that Apollon gene silenced by RNAi in combination with TMP intervention may reverse the drug resistance of leukemia cells.