放射诱导启动子介导双自杀基因治疗颊癌的实验研究

Radiation-inducible promoters-mediated cdglytk gene in the treatment of buccal carcinoma in golden hamster

目的观察放射诱导启动子介导胞嘧啶脱氨酶和单纯疱疹病毒胸苷激酶双融合自杀基因(CDglyTK)靶向治疗金黄地鼠颊癌的疗效。方法用二甲基苯并蒽(DMBA)诱导金黄地鼠颊黏膜癌变,建立颊癌动物模型,以脂质体为载体介导携放射诱导启动子调控双融合自杀基因的真核表达质粒 pcDNA3.1(+)/E-CDglyTK 瘤内转染并辅以3 Gy 放疗,腹腔注射5-氟胞嘧啶和丙氧鸟苷,描绘肿瘤生长曲线,RT-PCR 检测 CDglyTK 的 mRNA 水平,免疫组化检测细胞核增殖抗原的表达,原位末端标记法检测肿瘤细胞凋亡。结果放射诱导启动子介导的 CDglyTK 对肿瘤生长有明显抑制作用,RT-PCR 可检测到转染后肿瘤细胞 CDglyTK 的 mRNA 表达,诱导放疗增强了其表达水平,电泳条带灰度值从0.214±0.016上调至0.247±0.019(P<0.05);诱导放疗能显著提高疗效,凋亡指数从24.53%上升为31.22%(P<0.05),增殖指数由25.13%下降至20.07%(P<0.05)。结论放射诱导启动子可作为基因治疗分子开关,调节 CDglyTK 基因在金黄地鼠颊癌细胞中的靶向表达,低剂量放射性照射可显著提高其疗效。

Objective To observe the therapeutic effect of CDglyTK gene mediated by radiation-inducible promoters in the treatment of buccal carcinoma in Golden Hamster. Methods Animal models of buccal carcinoma in golden hamster were established by painting 0. 5% dimethyl-benzanthracene. The plasmids pcDNA（ ＋ ）3.1/E-CDglyTK were transfected into tumors by lipofectamine. 24 h later, the tumors were exposed to 3 Gy irradiation. Animals were monitored at regular intervals for volume of tumors. CDglyTK mRNA was assayed by RT-PCR. Apoptosis and proliferating cell nuclear antigen were detected respectively by in situ end-labeling and immunohistiochemical methods. Results Compared with control groups, the tumor was suppressed obviously by CDglyTK gene therapy combined with 3 Gy induction radiation. The expression of CDglyTK gene could be detected by RT-PCR in the transfected tumor, and up-regulation of CDglyTK expression was found in tumor exposed to radiation （ P 〈 0. 05 ）. There was significant difference in apoptosis index or proliferation index between tumor without irradiation and tumor with irradiation （P 〈 0. 05）. Conclusions The radiation-inducible promoter can be served as a molecular switch to regulate the expression of CDglyTK gene in buccal carcinoma in golden hamster, and low dose induction radiation can sianificantly improve the therapeutic effects.