人可溶性TRAIL蛋白诱导骨肉瘤细胞凋亡的实验研究  

Induction of apoptosis in MG-63 cell line by human soluble TRAIL

作　　者：祝少博[1] 陈振光[1] 喻爱喜[1] 方成[1] 

机构地区：[1]武汉大学中南医院显微骨科,430071

出　　处：《中华骨科杂志》2003年第6期339-344,共6页Chinese Journal of Orthopaedics

摘　　要：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 TNF-related apoptosis-inducing ligand(TRAIL)was a new member of TNF family.It could quickly inactivate tumor cell originated from different tissues but no effects on normal tissue in vitro.Osteosarcoma was the most common primary malignant bone tumor with the survival rate of 5years no more than20%after simple surgical management.The induced apoptosis and selective cytotoxicity of human soluble TRAIL for MG -63cell line were investigated in order to explore the feasibility of sTRAIL in clinical treatment of osteosarcoma.Methods sTRAIL-mediated cytotoxicity by MTT assay in MG-63osteosarcoma cell line,MRC-5human diploid lung cell line and L-02fetus hepar cell line were assessed,apoptotic cellular morphological transformation by phase contrast microscope and electron micro-scope was observed.The expression of TRAIL receptors mRNA in the cell lines was examined by RT -PCR.The apoptotic rates of MG-63cell line were shown by flow cytometry.Moreover,the apoptosis of MG-63cell line induced by sTRAIL was confirmed by terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL).Results MG-63,MRC-5and L-02cell lines were treated for 24hours with dif ferent concentrations of sTRAIL and the inhibitive rates of sTRAIL were evaluated with MTT assay,the in hibitive rates of 500ng /ml,1μg /ml ,2μg /ml and5μg /ml TRAIL inducing MG-63osteosarcoma cell line were10.1%,24.3%,50.6%and more than95%respectively.By flow cytometry,an obvious apoptosis peak ahead the diploid peak was obtained when MG-63cell line incubating with2μg/ml TRAIL for 6hours,howev er,the consequences of MRC-5and L-02cell line were non-differentiated.Therefore,MG-63cell line was significantly sensitive to sTRAIL-mediated apoptosis,but MRC-5and L-02cell line were resistant to sTRAIL-induced cell death.Under phase contrast microscope,when MG-63cell line were treated by sTRAIL,some cells began to became small and round in3to8hours.In24hours a large number of cells exfoliated and drifted in culture medium.Moreover,karyop

关 键 词：TRAIL 骨肉瘤细胞 凋亡 诱导 二倍体细胞 蛋白 肝细胞 抑制率 核膜 染色质 

分 类 号：R738.1[医药卫生—肿瘤]