RGD修饰的腺病毒介导LIF和IL-24双基因共表达对MEG01白血病细胞的抑制作用

Enhanced anti-tumor effect of RGD-modified adenovirus mediated LIF and IL-24 co-expression on MEG01 human leukemia cells

目的利用RGD修饰改造白血病抑制因子(LIF)和白细胞介素-24(IL-24)双基因共表达腺病毒载体,以提高感染效率,探讨其对人白血病MEG01细胞的抑制作用。方法同源重组法构建RGD修饰的表达LIF、IL-24的腺病毒载体Ad.RGD-LIF、Ad.RGD-IL24和Ad.RGD-LIF-IL24。在QBI-293A细胞中扩增腺病毒,检测病毒滴度。流式细胞仪检测各组腺病毒载体对MEG01细胞的感染效率。应用Western blotting检测目的基因LIF、IL-24在MEG01细胞中的表达。CCK-8法检测各组腺病毒载体Ad.RGD-LIF、Ad.RGD-IL24、Ad.RGD-LIF-IL24感染MEG01细胞后对细胞增殖的影响。PE-Anexin V/7-AAD双染后,经流式细胞仪检测细胞凋亡的变化。Western blotting检测凋亡相关蛋白的表达。PI染色后,流式细胞仪检测目的基因表达对MEG01细胞周期的影响。实时定量PCR检测细胞周期调控基因p21和E2F1的表达。结果成功构建了RGD修饰的腺病毒载体Ad.RGD-LIF、Ad.RGD-IL24和Ad.RGD-LIF-IL24。RGD修饰后的腺病毒能够显著增强对MEG01细胞的感染效率。CCK-8检测显示,与PBS组比较,携带单个目的基因的腺病毒载体Ad.RGD-LIF和Ad.RGD-IL24在第5 d对细胞生长的抑制率分别为29.2%和31.5%,均能够显著抑制MEG01细胞的生长;携带Ad.RGD-LIF-IL24双基因组的抑制率达42.5%,优于单基因组,差异均有统计学意义(P<0.05)。凋亡检测显示,与PBS组(4.5%)和空病毒组Ad.RGD(7.4%)比较,Ad.RGD-IL24(20.9%)、Ad.RGD-LIF(17.8%)均能够显著促进细胞凋亡,且Ad.RGD-LIF-IL24双基因组(29.7%)的促凋亡作用更强。Western blotting显示,LIF和IL-24能够提高促凋亡蛋白p53、Bax的表达,同时抑制抗凋亡蛋白Bcl-xl的表达。目的基因LIF、IL-24过表达会影响MEG01细胞周期的进程,使细胞阻滞在G2期。实时定量PCR显示,LIF和IL-24能够上调细胞周期调控基因p21的转录,抑制E2F1的表达。结论 LIF和IL-24过表达的腺病毒载体在体外通过调节p53、Bax、Bcl-xl的表达,影响白血病细胞MEG01的生长,诱导其凋亡,并通过调控p21、E2F1的水平影响细胞周期的进程。

Objective To construct RGD-modified leukemia inhibitory factor（ LIF） and interleukin-24（ IL-24） gene co-ex-pression adenovirus vector, and detect the inhibition effect for human leukemia MEG01 cells from LIF and IL-24. Methods The RGD-modified adenovirus Ad.RGD-LIF, Ad.RGD-IL24 and Ad.RGD-LIF-IL24 were construct by using the method of homologous re-combination. The adenovirus in QBI-293A cells were amplified, then the titer of adenovirus were detected. The infection efficiency of MEG01 leukemia cells were detected by flow cytometry. After infecting Ad.RGD-LIF, Ad.RGD-IL24 and Ad.RGD-LIF-IL24 adenovirus 48 hours, the expressions of LIF and IL-24 in MEG01 cells were detected by Western blotting method. The CCK-8 and PE-AnexinV/7-AAD method were used to detect the growth inhibition and apoptosis of MEG01 cells. Then p53, Bax and Bcl-xl gene expressions in＆amp;nbsp;MEG01 cells infected with adenovirus were detected by Western blotting method. PI staining method was used to detect the cell cycle of MEG01 cells. Real-time PCR method was used to detect p21 and E2F1 gene expressions. Results The RGD-modified adenovirus Ad. RGD-LIF, Ad.RGD-IL24 and Ad.RGD-LIF-IL24 were construct successfully. Compared with the Ad-GFP group, RGD-modified adeno-virus could enhance the infection efficiency of MEG01 cells significantly. Exogenous gene LIF and IL-24 could inhibit the growth of MEG01 cells and induce cell apoptosis through the regulation of p53, Bax and Bcl-xl gene expression. Besides, the double gene expres-sion has a synergistic effect. In MEG01 cells, LIF and IL-24 over-expression could induce cell cycle arrest. Furthermore, LIF and IL-24 could up-regulate the expression of cell cycle regulated gene p21 and inhibit the E2F1 expression. Conclusion LIF and IL-24 co-expression adenovirus can inhibit the MEG01 cell growth, induce cell apoptosis and cell cycle arrest, probably by regulating the expres-sion of p53, Bax and Bcl-xl in vitro.