沉默c-myc基因对Jiyoye细胞增殖与凋亡的影响

目的 运用RNA干扰（RNAi）技术，探讨经慢病毒载体介导的小干扰RNA（siRNA）转染Jiyoye细胞对肿瘤细胞增殖及凋亡的影响，以期为靶向c-myc基因的白血病和恶性淋巴瘤基因治疗提供体外实验依据。方法 设计、合成一对针对c-myc基因的c-myc-3寡聚核苷酸链和一对阴性对照c-myc- neg寡聚核苷酸链。退火，然后连接到pLVX干扰载体上，包装，慢病毒介导转染人Jiyoye细胞株。转染后培养72 h，流式细胞术检测各组细胞转染率，pLVX-c-myc-3转染Jiyoye细胞，MTT法检测细胞增殖；Annexin V-异硫氰酸荧光素/碘化吡啶（FITC/PI）双染流式细胞术检测细胞凋亡。结果 ①转染72 h后，流式细胞术鉴定阳性细胞比例，c-myc-neg组与c-myc-3组比较，差异无统计学意义（P 〉 0.05）（54.3 ± 4.2） % vs（52.8 ± 2.9） %]；两组细胞平均荧光强度差异、细胞转染率差异无统计学意义。②转染168 h生长曲线显示，转染pLVX-c-myc实验组细胞生长缓慢，与阴性对照组和空白对照组比较，差异有统计学意义（P 〈 0.05）。转染pLVX-c-myc 72 h后，实验组细胞抑制率为（46.40 ± 1.41） %，阴性对照组细胞抑制率为（17.03 ± 1.32） %，差异均有统计学意义（P 〈 0.05）。③转染pLVX-c-myc 72 h后，实验组早期凋亡细胞比例（25.6 ± 4.2） %，阴性对照组早期凋亡细胞（15.1 ± 4.2） %，空白对照组早期凋亡细胞为（12.7 ± 1.8） %，差异有统计学意义（P 〈 0.05）。实验组晚期凋亡细胞比例（11.5 ± 4.7） %，阴性对照组为（9.3 ± 4.7） %，空白对照组为（8.14 ± 2.8） %，差异有统计学意义（P 〈 0.05）。结论 设计合成构建的干扰序列pLVX-c-myc-3，可沉默c-myc基因，抑制Jiyoye细胞增殖，诱导细胞凋亡，为进一步探讨沉默c-myc基因在白血病和淋巴瘤靶向治疗中的作用提供了实验依据。

Effects of silencing c-myc gene by siRNA on Jiyoye cell growth and apoptosis

Objective To explore the effect of suppressing c-myc gene expression by lentiviral vector-based siRNA on the proliferation and apoptosis of Jiyoye cells, and provide experimental evidence for novel therapeutic approaches for leukemia and malignant lymphoma by targeting c-myc gene. Methods One short hairpin RNA （siRNA） interference sequence targeting c-myc gene （c-myc-3） and one negative control sequence （c-myc-neg） were designed and synthesized. They were annealed and cloned into pLVX vector to produce the recombinant plasmid, which was then transfeeted into Jiyoye cells by lentivirus. 72-hour after transfeetion, the transfection rate in each group was detected by flow eytometry（FCM）. The cell proliferation activities were assayed by MTT, and the apoptosis was analyzed by flow eytometry with Annexin V/PI double staining. Results ①72-hour after transfeetion, no significant difference in terms of positive cell rates and mean fluorescence intensity was observed between each group（P 〉 0.05）, indicated that the transfeetion efficiency of each group was not significantly different. ②During the 168 hours after transfeetion, the cells transfected with c-myc-3 all grew more slowly as compared with the cells in the negative control group and blank group, and the differences were statistically signifieant（P 〈 0.05）. 72-hour after being tranfected with e-myc-3, the cell inhibition ratio in the experimental group was significantly higher than that in the negative control group and blank control group（P 〈 0.05）. ③72-hour after transfection with pLVX-e-myc-3, the proportion of apoptotic cells in the experimental group was （25.6 ± 4.2） %, and that in the negative control group and blank control group was （15.1 ± 4.2） % and （12.7 ± 1.8） %, respectively. The proportion of early-term apoptotic cells in the experimental group was significantly higher than that in the negative control group and blank control group（P 〈 0.05）. The proportion of late-term apoptotic cells in the experimental group was （11.5 ± 4.7） %, and that in the negative control group and blank control group was （9.3 ± 4.7） % and （8.4 ± 2.8） %, respectively. The proportion of late-term apoptostie cells in the experimental group was significantly higher than that in the negative control group and blank control group（P 〈 0.05）. Conclusion It is demonstrated that the siRNA targeting c-myc inhibited tumor cell proliferation and induced cell apoptosis,which further provided an experimental basis for c-myc gene silencing in targeted therapy of leukemia and lymphoma.