重组腺病毒介导的突变型P27^(kip1)基因对胆管癌细胞化疗增敏机制的研究

Effect of adenovirus-mediated mutant exogenous P27^(kip1) gene expression on the chemosensitivities of cholangiocarcinoma cell line

目的探讨通过重组腺病毒转导突变型 P27^(kipl)至人胆管癌细胞系 QBC_(939)中,观察对胆管癌细胞化疗敏感性的影响并探讨可能的机制。方法重组腺病毒载体 Ad-P27mt 转染人胆管癌细胞系 QBC_(939),经逆转录-聚合酶链反应(RT-PCR)、Western blot 检测 P27^(kipl)在胆管癌细胞中的表达;流式细胞术检测细胞周期。通过噻唑蓝比色法及流式细胞仪检测5-氟尿嘧啶(5-FU)、丝裂霉素(MMC)、环磷酰胺(CTX)对转染 P27^(kipl)前后的 QBC_(939)细胞生长抑制及凋亡的影响。结果 Ad-P27mt 转染人胆管癌细胞系 QBC_(939)后,5-FU、MMC 和 CTX 对 QBC_(939)细胞生长抑制率分别由41.89%、45.59%和38.91%明显升高为56.15%、55.65%和51.69%;凋亡率也由13.76%±3.03%(5-FU)、11.76%±3.99%(MMC)和10.46%±2.10%(CTX)明显升高为41.39%±4.32%(5-FU)、35.94%±2.71%(MMC)和34.46%±2.32%(CTX),差异均有统计学意义(P<0.05)。结论突变型 P27^(kipl)能显著增强胆管癌细胞对化疗药物的敏感性,为靶向调控细胞周期联合化疗药物治疗胆管癌提供了参考依据。

Objective To investigate the effects of mutant exogenous P27^kip1 gene on chemosensitivity of human cholangiocarcinoma cell line. Methods The recombinant vector was constructed and the mutant P27^kip1 gene was transfected into human cholangiocarcinoma cell line （ QBC939 ）. RT-PCR and Western blot were used to determine the expression of target genes. The effects of 5-fluorouracil （5- FU）, mitomycin C （MMC） and cyclophosphamide （CTX） on the transfected cells were detected by assaying the apoptotic rate and growth inhibition by methabenzthiazuron （MTT） assay and flow cytometry （FCM）. Results The mutant exogenous P27^kip1 gene was expressed effectively in the cells, and the expression enhanced the apoptosis and growth inhibition of QBC939 inducted by 5-FU, MMC and CTX. The ratio of growth inhibiting increased significantly from 41.89% （5-FU）, 45.59% （ MMC）, 38.91% （CTX） to 56. 15% （5-FU）, 55.65% （ MMC）, 51.69% （ CTX）, and apoptosis index from 13.76% ± 3.03% （5-FU）, 11.76% ±3.99%（MMC）, 10.46% ±2. 10%（CTX） to41.39% ±4.32%（5-FU）, 35.94% ± 2.71%（MMC）, 34.46% ±2.32% （CTX） （P 〈0.05）. Conclusions The exogenous P27^kip1 gene transfer can remarkably increase the drug sensibility of the cholangiocarcinoma cells. The strategy targeted to control the cell cycle may be more effective in cancer treatment by combination of P27^kip1 gene therapy.