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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 investigate the inhibitory effect of liposome mediated plasmid pIRES-EGFP-tk containing TK gene on the growth of human osteosarcoma MG-63 cells induced by GCV combined with cisplatin (TK / GCV system). Methods The recombinant plasmid pIRES-EGFP-tk was constructed and transformed into competent E.coli BL21. The recombinant plasmid pIRES-EGFP-tk was identified and sequenced. The human osteosarcoma cell line MG-63 was cultured and transfected into human osteosarcoma cell line MG-63. Flow cytometry and fluorescence inverted microscopy were used to observe the cell growth. Stable cell lines and MTT assay were used to detect the inhibitory effect of GCV combined with different concentrations of cisplatin on the growth of osteosarcoma MG-63 cell line. Results The recombinant plasmid pIRES-EGFP-tk containing the cDNA fragment of TK gene was successfully constructed. The site and size of the plasmid were confirmed by PCR and restriction enzyme digestion. The recombinant plasmid pIRES-EGFP-tk transfected into human osteosarcoma MG-63 cell lines, stable expression. Under a fluorescence microscope MG-63 (tk +) showed green fluorescence. Flow cytometry detection of transfection rate was 67.7%. MTT assay showed that the growth of osteosarcoma cell line MG-63 (tk +) was inhibited when the GCV concentration was 10μg / ml, and the lowest effective concentration of cisplatin was 1μg / ml. When cisplatin was used in combination with GCV (5μg / ml), the effective concentration of cisplatin could be reduced to 0.25μg / ml, and its inhibitory effect on MG-63 (tk +) cell line was obviously inhibited. Conclusion TK / GCV combined with cisplatin can inhibit the growth of human osteosarcoma MG-63 cells and increase the sensitivity of MG-63 cells to cisplatin, and reduce its effective dose. It is expected to provide experimental evidence for the adjuvant treatment of osteosarcoma.