弓形虫诱导人白血病细胞K562凋亡的实验观察

目的 探讨弓形虫对体外培养的人白血病细胞K562（简称K562细胞）有无抑制作用和诱导细胞凋亡作用。方法 取培养至对数生长期的K562细胞（浓度为5×10^-4/ml和1×10^-6/ml）分别接种于96孔培养板（100 μl/孔）及50 ml培养瓶（1.5 ml/瓶）中，加入不同浓度的弓形虫速殖子（1×10⁴、2×10⁴、4×10⁴、8×10⁴及16×10⁴个/ml），作用48 h, 用四甲基氮噻唑蓝（MTT）法检测其对K562细胞增殖的抑制作用；荧光显微镜、琼脂糖凝胶电泳及流式细胞仪检测细胞凋亡。 结果 上述不同浓度弓形虫速殖子作用48 h，对K562细胞增殖的抑制率依次为17%、28%、48%、50%及55%。各实验组吸光度（A₄₉₀）与对照组比较差异均有统计学意义（t=3.606及5.918， P<0.05； t=9.171、7.841、7.067， P<0.01）。荧光显微镜观察实验组培养的K562细胞，出现细胞核固缩及凋亡小体。琼脂糖凝胶电泳见DNA梯形条带。流式细胞仪检测到细胞凋亡峰（48 h），上述不同浓度弓形虫速殖子诱导K562细胞凋亡数分别占总数的5.53%、7.12%、10.34%、21.14% 及29.68%。对照组未出现凋亡小体。 结论 弓形虫速殖子对体外培养K562细胞增殖有明显的抑制作用，并可诱导K562细胞凋亡。

Apoptosis of Human Leukemia K562 Cell in vitroInduced by Toxoplasma gondii

OBJECTIVE: To investigate whether the Toxoplasma gondii can inhibit proliferation of human leukemia K562 cells and/or induce apoptosis of the cells in vitro. Methods K562 cells (5x10(4)/ml) were harvested at mid-exponential phase and planted in 96 well plates with 100 microl each and in 50 ml culture bottles, 1.5 ml each. The cells were treated for 48 hours with different concentration of Toxoplasma tachyzoites. Growth inhibition rate was measured with MTT method. Apoptosis was detected through following ways: fluorescence microscopy with Hoechst 33 258 staining was used for observing the change of cell morphology, agarose electrophoresis was used to detect the DNA changes and FCM was used to observe sub-diploid. RESULTS: Toxoplasma can inhibit proliferation of K562 cells. K562 cells treated with Toxoplasma presented an inhibition rate of 17%, 28%, 48%, 50% and 55% under the tachyzoite concentration of lxl0(4), 2x10(4), 4x 10(4), 8x10(4) and 16xl0(4)/ml respectively, with a significant difference to the control (t=3.606, 5.918, P<0.05; t=9.171, 7.841 and 7.067, P<0.01). Cell contraction and apoptotic bodies were observed under fluorescence microscope. DNA fragment was shown through agarose electrophoresis. Flow cytometric analysis showed an apoptosis peak at 48h. The apoptosis rate was 5.53%, 7.12%, 10.34%, 21.14% and 29.68% respectively. CONCLUSION: Toxoplasma gondii inhibits proliferation and induces cell apoptosis in K562 cells in vitro.