Apollon siRNA对慢性髓系白血病K562细胞株阿糖胞苷敏感性影响研究

目的：研究小干扰RNA（RNAi）技术沉默凋亡抑制蛋白Apollon基因联合小剂量阿糖胞苷（Ara-C）对K562细胞化疗敏感性的影响。方法：构建pGPHI-GFP-Neo-Apollon真核表达载体,经阳离子脂质体转染K562细胞,实验分为RNAi组、阴性质粒组、细胞对照组、Ara-C组和RNAi＋Ara-C组5组,采用RT-PCR和细胞免疫荧光检测干扰效果。RNAi与小剂量Ara-C单独和联合应用后,采用MTT法检测细胞增殖能力,流式细胞术检测细胞凋亡率。结果：RT-PCR结果显示,实验组Apollon mRNA的相对表达量为（27.622±0.057）%,与细胞对照组（89.770±0.028）%和阴性对照组（84.868±0.057）%相比明显降低,差异有统计学意义,F=57.573,P=0.012。细胞免疫荧光检测结果显示,实验组细胞Apollon蛋白的荧光强度为（15.96±1.71）%,明显低于细胞对照组（35.36±2.90）%和阴性对照组（34.97±2.65）%,差异有统计学意义,F=71.063,P=0.013。重组载体和（或）10μg/mL Ara-C作用于K562细胞24、48和72h后,RNAi组和RNAi＋Ara-C组K562细胞增殖抑制率（IR%）明显增高,随着作用时间的延长,抑制效果越明显。流式细胞术结果显示,RNAi＋Ara-C组细胞凋亡率为（30.816±1.06）%,明显高于细胞对照组（4.803±0.112）%、阴性对照组（4.566±0.205）%和Ara-C组（11.61±0.604）%及RNAi组（20.226±0.986）%,差异有统计学意义,F=138.57,P〈0.001。结论：靶向Apollon RNAi联合小剂量Ara-C能有效抑制K562细胞增殖并促进其凋亡,显著减少Ara-C使用剂量,明显提高了K562细胞对Ara-C的敏感性。

Apollon siRNA of Chronic Myeloid leukemia K562 cell line to cytarabine drugs sensitivity research

OBJECTIVE： To study if RNA interference （RNAi） technology Apollon,silence apoptosis inhibiting pro- tein gene,in combination with low dose cytarabine （Ara-C） can improve the K562 cells sensitivity to chemotherapeutic drugs. METHODS： pGPHI-GFP-Neo-Apollon eukaryotic expression vector was built and transfected into K562 cells by positive ion liposome. The experiment was divided into five groups： RNAi group, negative group, normal control group, Ara-C group,RNAi＋ Ara-C group. RNAi single and combined with low dose of Ara-C after application was determined by MTT method to detect cell proliferation and {low cytometry was used to detect cell apoptosis rate. RESULTS： RT-PCR showed that the Apollon mRNA expression level of the experimental group（27. 622± 0. 057）% was lower significantly compared with expression level of cell control group（89. 770±0. 028）% and control group（84. 868±0. 057） % ,the differ- ence was statistically significant （F= 57. 573,P= 0. 012）. Cell immunofluorescence results showed that the Apollon pro- tein expression level （15. 96± 1. 71）% was lower significantly compared with expression level of cell control group （35.36±2. 90）% and control group （34. 97 ±2. 65）%, the difference was statistically significant （F = 71. 063, P = 0. 013）. After PGPHI-GFP-Neo-Apollon carrier in combination with small dose of Ara-C（10 μg/mL） effected on K562 cell for 24,48 and 72 h, RNAigroup and RNAi＋ Ara-Cgroup cell proliferation capacity decreased obviously, and the inhibi- tion effect was time-dependent. FCM showed that RNAi＋Ara-C group Ccell apoptosis rate was （30. 816± 1.06） % signif- icantly higher than that of control group （4. 803±0. 112） % ,negative control group （4. 566±0. 205）% and Ara-C group （11.61±0. 604）% and RNAi group （20. 226±0. 986）% ,the difference was statistically significant among the 5 groups （F= 138.57, P〈0. 001）. CONCLUSION：Targeting of RNAi in combination with low dose of Ara-C Apollon could inhib