双启动子引导自杀基因靶向杀伤5-FU耐药肿瘤细胞的研究

目的：探讨胸苷酸合成酶（TS）基因启动子和p16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、p16双启动子重组表达载体,将TK基因插入TS、p16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,观察双启动了引导TK基因特异杀伤肿瘤细胞的作用。结果：将TS、p16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：9／300、92／300。转染组瘤细胞集落形成率明显降低（t=33.885,P＜0．01）,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,p16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p16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。

Double promoters induct suicide gene target killing of 5-FU drug-fast cancer cells

OBJECTIVE: To study target killing of 5-FU drug-fast cancer cells with thymidylate synthase (TS) and p16 gene promoters inducting TK gene expression. METHODS: TS promoter was inserted to 5' end and p16 promoter inserted to 3' end of TK cDNA sequence, constructing recombinant plasmid of pXJ41. Human rectal cancer cell lines of HR-8348 and normal peripheral blood mononuclear cells (PBMC) were transfected with the recombinant plasmid. Plating efficiency was counted and survival rates of cells were tested with MTT method. And suppression rates of xenograft tumors in nude mice were examined. RESULTS: Recombinant pXJ41 with double promotors and TK gene was transfected into HR-8348, and positive expression of TS and TK was observed. The expression of TK gene was consistent with TS expression. Plating efficiency was 9/300, 92/300 in transfected HR-8348 and contrast cells respectively (t = 33.885, P < 0.01). Cancer cell growth rate reduced markedly in the transfected group. The suppression rate of xenograft tumor growth was 74.5%. With the recombinant pXJ41 to transfect PBMC, p16 expression was positive, but TK and TS expressions were negative. No damnification was observed in PBMC. CONCLUSIONS: TS and p16 double promoters are capable of inducting TK target killing of 5-FU drug-fast cancer cells, thus protecting normal cells and improving safety of gene therapy.