TK/GCV系统联合顺铂对骨肉瘤MG-63细胞生长的抑制作用研究

A study on the inhibitory effects of thymidine kinase-ganciclovir system combined with cisplatin on the growth of osteosarcoma MG-63 cells

目的研究脂质体介导的包含TK基因的质粒pIRES-EGFP-tk在GCV作用下(TK/GCV系统)联合化疗药物顺铂对人骨肉瘤MG-63细胞生长的抑制作用。方法构建重组质粒pIRES-EGFP-tk,制备和转化感受态大肠杆菌BL21及鉴定测序,培养和转染人骨肉瘤MG-63细胞株,流式细胞仪分析和荧光倒置显微镜下观察细胞生长状态,筛选稳定细胞系,MTT法检测GCV与不同浓度顺铂联合应用对骨肉瘤MG-63细胞株生长的抑制作用。结果成功构建含TK基因cDNA片段的重组质粒pIRES-EGFP-tk,通过PCR、酶切电泳等方法鉴定质粒位点和大小均符合实验设计。将重组质粒pIRES-EGFP-tk转染入人骨肉瘤MG-63细胞株,获得稳定表达。荧光显微镜下观察MG-63(tk+)呈现绿色荧光。流式细胞仪检测转染率为67.7%。通过MTT法检测,当GCV浓度为10μg/ml时,骨肉瘤MG-63(tk+)细胞株的生长受到抑制,同时测得顺铂的最低有效浓度为1μg/ml。当顺铂与GCV(5μg/m1)联合应用时,顺铂的有效浓度可降为0.25μg/ml,并对MG-63(tk+)细胞株有明显的抑制作用。结论 TK/GCV系统联合顺铂可对人骨肉瘤MG-63细胞的生长产生抑制作用,并提高MG-63细胞对顺铂的敏感性,降低其有效剂量。可望为骨肉瘤的辅助治疗提供实验依据。

Objective To study the inhibitory effects of the liposome–mediated plasmid （pIRES-EGFP-tk） containing thymidine kinase （TK） gene under the action of ganciclovir （GCV） （TK/GCV system） combined with the chemotherapy drug cisplatin （cDDP） on the growth of osteosarcoma MG-63 cells. Methods The recombinant plasmid pIRES-EGFP-tk was constructed, and the Escherichia coli BL21 competent cells were prepared and transferred. Sequencing was identified. The osteosarcoma MG-63 cell lines were cultured and transfected. The cell growth state was analyzed and observed by flow cytometry and under inverted fluorescence microscopy. The steady cell lines were screened, and the inhibitory effects of GCV combined with various concentrations of cDDP on the growth of osteosarcoma MG-63 cell lines were evaluated by 3-（4,5）-dimethylthiahiazo （-z-y1）-3,5-di- phenytetrazoliumromide （MTT） assay. Results The recombinant plasmid pIRES-EGFP-tk of the complementary deoxyribonucleic acid （cDNA） fragment containing the TK gene was successfully constructed. By such methods as polymerase chain reaction （PCR）, enzyme electrophoresis and so on, the position and size of the plasmid were identified, and the results were consistent with the experimental design. The recombinant plasmid pIRES-EGFP-tk was transfected into the osteosarcoma MG-63 cell lines, with steady expressions. The MG-63 （tk＋） cells were observed to show green fluorescence under fluorescence microscope. The transfection efficiency was 67.7%, which was tested by flow cytometry. The osteosarcoma MG-63 （tk＋） cell line growth was repressed by GCV with the concentration of 10 μg/ml by MTT assay, and while the minimal effective concentration of cDDP was 1 μg/ml. When GCV （5μg/ml） was combined with cDDP, the minimal effective concentration of cDDP was reduced to 0.25 μg/ml, which still had significant inhibitory effects on the MG-63 （tk＋） cell lines. Conclusions The TK/GCV system combined with cDDP can repress the growth of osteosarcoma MG-63 cells, and enhance the sensibility of MG-63 cells to cDDP, and further to reduce the effective dose. It is expected to provide experimental evidence for the adjuvant therapy for osteosarcoma.