VP_3诱导骨肉瘤多药耐药细胞凋亡作用机制的初步研究

A preliminary study on the mechanism of VP_3 inducing apoptosis in the multidrug-resistant model of human osteosarcoma cell line

目的 研究VP3 对人成骨肉瘤细胞OS 73 2多药耐药株R OS 73 2治疗作用的分子作用机制与途径。方法 VP3 基因经酶切后克隆入质粒 pIRES1,获得重组质粒pIRVP3 。脂质体法 pIRVP3 转染R OS 73 2细胞 ,RT PCR检测VP3 在细胞中的表达。提取瘤细胞基因组DNA进行琼脂糖凝胶电泳。流式细胞仪检测凋亡率。Western blot检测p5 3、bcl 2蛋白表达。结果 VP3 基因已在转录水平表达。电泳见转染组DNA呈梯状条带 ,对照组为单一条带。转染组凋亡率明显高于空白对照组 (P <0 .0 1)。转染组与对照组 p5 3及bcl 2蛋白显色带灰度无明显差异。 结论 VP3 可有效诱导R OS 73 2细胞凋亡 ,其分子作用机制与途径可能不依赖 p5 3及bcl 2作用。

ObjectiveTo study the mechanism of VP 3 inducing apoptosis in the multidrug-resistant model of human osteosarcoma cell line (R-OS-732).Methods363 bp VP 3 gene was cloned into the vector pIRES1 and the recombinant eukaryon expression vector pIRVP 3 was transfected into R-OS-732 cells with liposome.The expression of VP 3 gene at transcription level was proved by RT-PCR.The transfected R-OS-732 cells were collected after they were cultivated 48 hours.The genomic DNA extracted from the cells were observed by agarose gel electrophoresis.The apoptosis rate of the cells was analysed by flow cytometry.The expression of p53 and bcl-2 was determined by Western-blot.ResultsThe transfected R-OS-732 cells were collected after they were cultivated 48 hours,many typical apoptosis cells in the transfected group were observed under electronmicroscope,DNA ladder of them was observed through agarose gel electrophoresis,and only one band was observed in the control group.Flow cytometry analysis showed that the apoptosis rate of the cells in the transfected group was relatively higher than that in the control group(P<0.01).There was no difference in the expression of p53 and bcl-2 of the two groups.ConclusionApoptin can induce apoptosis in the multidurg-resistant model of human osteosarcoma cell line(R-OS-732) without p53 and bcl-2.