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目的初步观察并探讨人重组可溶性TRAIL蛋白诱导MG-63骨肉瘤细胞凋亡的作用,并通过实验初步探讨TRAIL对肿瘤细胞的选择性杀伤作用。方法MTT法测定TRAIL对MG-63细胞、MRC-5人肺二倍体细胞及L-02人肝细胞的抑制率;电镜观察与FACS分析TRAIL蛋白诱导MG-63骨肉瘤细胞凋亡的作用;用RT-PCR检测TRAIL受体在MG-63骨肉瘤细胞、MRC-5人肺二倍体细胞及L-02人肝细胞上的表达,并初步分析TRAIL诱导MG-63骨肉瘤细胞凋亡的机制。结果MTT还原试验表明:作用24h后500ng/mlTRAIL的抑制率为10.1%,1μg/mlTRAIL的抑制率为24.3%,2μg/mlTRAIL的抑制率为50.6%,5μg/mlTRAIL的抑制率为95%以上,其半数抑制浓度为2μg/ml。而TRAIL对MRC-5人肺二倍体细胞株及L-02人肝细胞株无明显作用。细胞形态学观察显示:MG-63骨肉瘤细胞经TRAIL作用3～8h后即有部分细胞开始变小、变圆,24h后大量细胞变圆、脱落,漂浮于培养液中。透射电镜可观察到核固缩,染色质浓聚于核膜形成新月状小体。流式细胞仪检测显示2μg/ml的TRAIL处理MG-63骨肉瘤细胞6h,即可在二倍体峰前出现较明显的凋亡峰。结论人可溶性TRAIL蛋白可以迅速地选择性杀伤MG-63骨肉瘤细胞,具有潜在的临床应用价值。
Objective To observe and explore the effect of human recombinant soluble TRAIL protein on MG-63 osteosarcoma cell apoptosis, and to explore the selective killing effect of TRAIL on tumor cells through experiments. Methods The inhibitory rate of TRAIL on MG-63 cells, MRC-5 human lung diploid cells and L-02 human hepatocytes was determined by MTT assay. The effect of TRAIL on MG-63 osteosarcoma cell apoptosis was analyzed by electron microscope and FACS. The expression of TRAIL receptor on MG-63 osteosarcoma cells, MRC-5 human lung diploid cells and L-02 human hepatocytes was detected by RT-PCR. The apoptosis of MG-63 osteosarcoma cells was detected by TRAIL mechanism. Results MTT reduction assay showed that the inhibitory rate of TRAIL at 500ng / ml was 10.1% after 24 h, the inhibition rate of TRAIL at 1μg / ml was 24.3%, the inhibition rate of TRAIL at 2μg / ml was 50.6%, the TRAIL at 5μg / ml was above 95% The half inhibitory concentration was 2 μg / ml. However, TRAIL had no significant effect on MRC-5 human lung diploid cell line and L-02 human hepatocyte cell line. Morphological observation showed that some cells started to become smaller and round after 3 ~ 8h of MG-63 osteosarcoma cells. After 24 hours, a large number of cells became round, shed and floated in the culture medium. Transmission electron microscopy can be observed nuclear pyknosis, chromatin condensation in the nuclear membrane to form crescentic bodies. Flow cytometry showed that 2μg / ml TRAIL treatment of MG-63 osteosarcoma cells 6h, peak appeared in the diploid before the more obvious peak. Conclusion Human soluble TRAIL protein can selectively kill MG-63 osteosarcoma cells rapidly and has potential clinical value.