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目的 探讨胸苷酸合成酶 (TS)基因启动子和p1 6基因启动子引导胸苷激酶 (TK)自杀基因靶向杀伤 5 FU耐药肿瘤细胞的作用。 方法 构建TS、p1 6双启动子重组表达载体 ,将TK基因插入TS、p1 6启动子之间 ,转染耐药人直肠癌细胞系HR 8348、外周血单个核细胞。通过克隆形成实验、细胞存活率测定和裸鼠移植瘤治疗实验 ,观察双启动子引导TK基因特异杀伤肿瘤细胞的作用。结果 将TS、p1 6双启动子重组质粒载体转入耐药HR 8348细胞 ,检测TS、TK基因表达阳性 ,TK基因与TS表达一致。转染组和对照组肿瘤细胞集落形成分别为 :9/ 30 0、92 / 30 0。转染组瘤细胞集落形成率明显降低 (t =33 885 ,P <0 0 1 ) ,癌细胞生长抑制率显著提高。对裸鼠移植瘤的生长抑制率为74 5 %。在转染的外周血单个核细胞中 ,p1 6表达阳性 ,TS、TK表达阴性。转染双启动子重组质粒对正常外周血单个核细胞无损伤作用。 结论 TS和p1 6双启动子可引导TK基因靶向性杀伤 5 FU耐药肿瘤细胞 ,保护肌体正常细胞 ,提高自杀基因治疗的安全性
Objective To investigate the role of thymidylate synthase (TS) gene promoter and p1 6 gene promoter in targeting 5-FU-resistant tumor cells targeting thymidine kinase (TK) suicide gene. Methods The recombinant expression vector of TS and p1 6 double promoters was constructed. The TK gene was inserted between TS and p1 6 promoters and transfected into drug-resistant human rectal cancer cell line HR 8348 and peripheral blood mononuclear cells. Through the experiment of clonality, cell viability and transplanted tumor in nude mice, the double promoter was used to guide the specific killing of tumor cells by TK gene. Results The TS and p1 6 double-promoter recombinant plasmids were transfected into drug-resistant HR 8348 cells. The positive expression of TS and TK was detected, and the expression of TK and TS were consistent. The colony formation of tumor cells in the transfected group and the control group were 9/30, 92/30, respectively. The rate of tumor colony formation in the transfection group was significantly lower (t = 33 885, P <0.01), and the growth inhibition rate of cancer cells was significantly increased. Growth inhibition rate of transplanted tumor in nude mice was 74.5%. Transfection of peripheral blood mononuclear cells, p1 6 expression was positive, TS, TK negative expression. Transfection of double promoter recombinant plasmid had no effect on normal peripheral blood mononuclear cells. Conclusion The TS and p16 double promoters can guide the TK gene to target 5 FU-resistant tumor cells, protect the normal cells and enhance the safety of suicide gene therapy