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∗

摘要

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⁃AnexinV／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 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.