TK基因转染骨肉瘤U-2细胞体外实验研究

目的 研究脂质体介导的TK/GCV系统对人骨肉瘤细胞的杀伤作用及其产生的旁观者效应.方法 构建重组质粒pIRES-EGFP-tk,制备和转化感受态大肠杆菌BL21以及鉴定测序,提取、纯化重组质粒DNA,培养和转染人骨肉瘤细胞株U-2 OS,流式细胞仪分析和荧光倒置显微镜下观察细胞生长状态,用转染后的培养液的上清加入TK （-） U-2 OS中观察旁观者效应.结果 成功地构建出包含有TK基因cDNA片段的重组质粒pIRES-EGFP-tk,通过PCR等方法鉴定质粒大小和位点均符合实验设计.荧光显微镜下观察U-2 OS （TK＋）呈现绿色荧光.U-2 OS TK/GCV培养上清液可明显诱导细胞凋亡.但将上清液用0.2μm的滤膜滤过后,诱导凋亡作用消失.结论成功构建重组质粒pIRES-EGFP-tk,体外实验表明人骨肉瘤细胞系U-2 OS对脂质体介导的TK/GCV系统敏感,并且有明显的旁观者效应,可望成为骨肉瘤基因治疗的新途径.

The experimental research on the osteosarcoma U-2 cells transfected by thymidine kinase gene in vitro

Objective To study the killing effects of the liposome-mediated thymidine kinase （TK）/ ganciclovir （GCV） system on human osteosarcoma cells and its bystander effects. Methods The recombinant plasmid pIRES-EGFP-tk was constructed, and then was prepared and transformed into Escherichia coli BL21 competent cells. And the sequencing was identified. The recombinant plasmid deoxyribonucleic acid （DNA） was extracted and purified, and the human osteosarcoma cell line U-2 osteosarcoma （OS） was cultured and transfected. The cell growth state was analyzed by flow cytometry and observed under inverted fluorescence microscope. After transfection, the supernatant of culture medium was added to the TK （-） U-2OS, and then its bystander effects were observed. Results The recombinant plasmid plRES-EGFP-tk containing complementary deoxyribonucleic acid （cDNA） fragments with TK gene was successfully constructed. By such method as polymerase chain reaction （PCR） and so on, the size and position of plasmids were identified, and the results were consistent with the experimental design. Under fluorescence microscope, U-2 OS （TK＋） was observed to show green fluorescence. The supernatant of culture medium of U-2 OS TK/GCV system had a significant effect of inducing apoptosis. However, when the supernatant was filtrated with 0.2μm filter membrane, its effects disappeared. Conclusions The recombinant plasmid plRES-EGFP-tk is successfully constructed. The experiment in vitro demonstrated that the human osteosarcoma cell line U-2OS is sensitive to the liposome-mediated TK/GCV system and its bystander effects are significant. It is expected to become a new gene therapy approach for the osteosarcoma.