IFN-γ基因修饰的骨髓基质细胞体外抗白血病作用的实验研究

In vitro anti-leukemic effects of bone marrow stromal cells modified with Ad/hIFN-γ

目的研究重组人IFNγ腺病毒(Ad/hIFNγ)在CML病人骨髓基质细胞(BMSCs)中的转染效率、IFNγ表达,并进一步观察转染Ad/hIFNγ的骨髓基质细胞体外抗白血病细胞株K562增殖和诱导K562细胞凋亡的作用。方法以Ad/hIFNγ重组腺病毒按感染复数(MOI)为50转染CML病人骨髓基质细胞,RTPCR、ELISA及Westernblot分别检测转染Ad/hIFNγ的人骨髓基质细胞(BMSCs)中IFNγmRNA和IFNγ的表达;转染Ad/hIFNγ的骨髓基质细胞与K562细胞共培养,通过绘制K562细胞生长曲线分析其对K562细胞增殖的影响;并进一步通过流式细胞学分析其对K562细胞周期的影响和诱导凋亡的作用。结果RTPCR、Westernblot及ELISA方法都分别检测到转染Ad/hIFNγ的人骨髓基质细胞(BMSCs)中IFNγmRNA和IFNγ的表达;与对照组相比,转染Ad/hIFNγ的人骨髓基质细胞的实验组具有明显的抑制K562细胞增殖的作用(P=0.000),在细胞周期G1期的K562细胞比例明显增加,而S期K562细胞比例显著下降(P<0.01),并显著诱导K562细胞的凋亡(P<0.01)。结论Ad/hIFNγ转染人骨髓基质细胞后可获得IFNγ有效的表达,并在体外能有效地发挥抗白血病细胞作用。

Objective This study was designed to explore the efficiency of recombinant adenovirus-mediated human Gamma Interferon (hIFN-γ) gene transferred into BMSCs and the expression of IFN-γ in vitro. We further studied the anti-proliferation and induction of apoptosis effects on the human leukemia cell line K562 cells by Ad/hIFN-γ transduced BMSCs in vitro. Methods On transduction of BMSCs by Ad/hIFN-γ at a MOI of 50, we detected the expression of IFN-γ mRNA and IFN-γ by RT-PCR, cell immunohistochemical staining, ELISA and Western blot. After incubation of K562 cells with Ad/hIFN-γ transduced BMSCs, The cellular proliferation of K562 cells was determined by the growth curve and MTT assay. Apoptosis and cell cycle status of K562 cells were analyzed by flow cytometry. Results We can detect the expression of IFN-γ mRNA and IFN-γ by RT-PCR, cell immunohistochemical staining, ELISA and Western blot. Ad/hIFN-γ transduced BMSCs markedly inhibited the proliferation of K562 cells compared with control groups after incubation with K562 cells (P=0.000). As compared with control groups, the percentage of K562 cells in the G1 phase of cell cycle can be increased by treatment of hIFN-γ produced by Ad/hIFN-γ transduced BMSCs, but the percentage of K562 cells in the S phase of cell cycle can be decreased in the same time (P<0.01, student t-test). The apoptosis effects can be induced by Ad/hIFN-γ transduced BMSCs (P<0.01, student t-test). Conclusion on transduction of BMSCs by Ad/hIFN-γ, IFN-γ expression can be acquired and local production of IFN-γ showed anti-leukemic effects in vitro.