重组人p53腺病毒注射液(今又生)对人肺腺癌细胞生长及化疗敏感性研究

Effect of recombinant adenovirus-p53 on growth and chemosensitivity of human lung adenocarcinoma cell lines

背景与目的在肺癌中,p53突变是最常见的基因改变之一,p53基因的突变常导致细胞对化疗不敏感。有研究表明导入野生型p53基因能增加化疗药物的敏感性。本研究的目的是探讨重组人p53腺病毒注射液(Adp53,今又生,Gendicine)对人肺腺癌细胞生长及化疗敏感性的影响。方法将重组腺病毒载体所携带的野生型p53基因导入人肺腺癌细胞株GLC82(含突变型p53基因)及A549(含野生型p53基因),并联合应用化疗药物顺铂(DDP),通过Westernblot法分析外源野生型p53基因在细胞内的表达,MTT法和流式细胞术观察对细胞生长及细胞周期、凋亡的影响。结果通过Westernblot证实了外源p53基因能在GLC82及A549细胞中高效表达。MTT法观察到Adp53对肺癌细胞的抑制作用呈时间依赖性和剂量依赖性效应。100MOIAdp53与0.5mg/LDDP联合应用后72h,对A549细胞的生长抑制率达43.13%±0.72%,显著高于单用Adp53组(23.44%±0.54%,P<0.001)和DDP组(14.17%±1.39%,P<0.001);对GLC82细胞生长的抑制率达63.73%±0.92%,显著高于单用Adp53组(41.51%±0.59%,P<0.001)和DDP组(56.11%±1.12%,P<0.001)。流式细胞术检测结果显示,Adp53与DDP联合应用能使细胞阻滞于G0G1期,S期细胞比例明显减少。Adp53+DDP组A549细胞凋亡率为28.99%±1.07%,显著高于单用Adp53组(15.35%±1.31%,P<0.001)和DDP组(1.74%±0.77%,P<0.001);Adp53+DDP组GLC82细胞凋亡率为62.98%±2.43%,显著高于单用Adp53组(20.88%±0.71%,P<0.001)和DDP组(6.91%±1.52%,P<0.001)。结论重组人p53腺病毒注射液能抑制肺腺癌细胞的生长,并不受内源性p53状态的影响。它与抗癌药DDP联用能显著增加肺腺癌细胞的化疗敏感性。

Baclkground and objective p53 gene is the most commonly mutated gene in lung cancer, p53 mutation results in insensitivity of cells when exposed to chemotherapy. It has been reported that adenovirus-mediated wild-type p53 gene transfection into lung cancer cells can enhance the cytotoxic effect of anti-cancer drugs. The aim of this study is to evaluate the effects of domestic recombinant adenovirus-p53 （Ad-p53, Gendicine） on growth and chemosensitivity of human lung adenocarcinoma cell lines. Methods Human lung adenocarcinoma cell lines GLC-82 （including mutant p53） and A549 （including wild-type p53） were treated with Ad-p53, cisplatin （DDP） or Ad-p53＋DDP respectively, p53 expression was detected by Western blot. The cell growth inhibition was assessed by MTT, and cell cycle and apoptosis were detected by flow cytometry. Results High-level p53 expression was detected in Ad-p53 infected GLC-82 and A549 cells by Western blot. There was a dose-dependent and time-dependent inhibition of cell proliferation by Ad-p53. After combined treatment with Ad-p53 （100 MOI） and DDP （0.5 mg/L） for 72 h, the growth inhibition rate of A549 cells was 43.13% ±0.72%, which was significantly higher than that in Ad-p53 group （23.44 %± 0. 54 %, P〈0. 001 ） and DDP group （14.17% ± 1.39 %, P〈0. 001） ; and the growth inhibition rate of GLC-82 cells was 63.73 % ±0. 920/6, which was significantly higher than that in Ad-p53 group （41.51%±0.59%, P〈0.001） and DDP group （56. 11% ± 1. 12%, P〈0. 001）. Combined administration of Ad-p53 and DDP remarkably arrested A549 and GLC-82 cells in G0-G1 , and cells in S phase significantly decreased. Meanwhile the apoptotic rate of A549 cells was 28.99 % ± 1.07 % in Ad-p53 ± DDP group, which was significantly higher than that in Ad-p53 group （ 15.35 % ± 1.31%, P〈0. 001 ） and DDP group （ 1.74 % ± 0.77 %, P〈0. 001）. The apoptotic rate of GLC-82 cells was 62.98% ±2.43% in Ad-p53＋DDP group, which was significantly higher than that in Ad-p53 group （20. 88%±0. 71%, P〈0. 001） and DDP group （6.91% ±1.52%, P〈0. 001）. Conclusion Ad-p53 （Gendicine） can inhibit the growth of human lung adenocarcinoma cell lines irrespective of the status of endogenous p53 gene. Its combination with DDP may significantly enhance the chemosensitivity of human lung adenocarcinoma cells to DDP.