腺病毒介导p27mt基因转移诱导恶性黑素瘤细胞凋亡的实验研究

Apoptosis of human malignant melanoma cells induced by recombinant adenoviral vector-mediated gene transfer of human p27mt

目的:探讨突变型p27基因(p27mutant type gene;p27mt)对恶性黑素瘤A375细胞凋亡的调节作用。方法:利用重组腺病毒Ad-p27mt转染培养的人恶性黑素瘤A375细胞,用3H-TdR掺入法检测细胞增殖;流式细胞术、DNA片段分析法、TUNEL法检测细胞凋亡。结果:重组腺病毒Ad-p27mt在MOI≥50时,可达到100%的转导效率。Ad-p27mt转染恶性黑素瘤细胞A375后,3H-TdR掺入法检测发现细胞增殖抑制;流式细胞术检测在G1期前出现亚二倍体凋亡峰;细胞DNA抽提电泳后发现凋亡特征性梯带。TUNEL法检测凋亡指数分别为48.5±3.6(Ad-p27mt组)及4.2±0.8(空白对照组),差异有显著性(P<0.01)。结论:重组腺病毒介导的p27mt基因转移可诱导恶性黑素瘤A375细胞在Gl期阻滞并导致细胞凋亡。

Objective： To investigate the effects of p27 mutant type gene on modulation of apoptosis of malignant melanoma cells A375. Methods： A replication of deficient adenovirus vector encoding p27mt （ad- p27mt）was used, and then p27mt cDNA was transferred into human malignant melanoma A375 cell lines in vitro. The synthesis of DNA in A375 cells was determined by using ^3H - thymidine incorporation. The cell apoptosis was determined by flow cytometry, TUNEL technique and DNA fragmentation analysis. Results： The virus titer was 1.26 × 10^12 cfu/ mL. The transduetion efficiency was 100% when multiplicity of infection ≥50. Flow cytometry analysis revealed a sub- G1 cell peak in ad - p27mt transdueted malignant melanoma cell lines. Agarose electrophoresis showed marked ladder characteristic of apoptosis. Meanwhile, ^3 H - thymidine ineorporation demonstrated that the synthesis of DNA in A375 cells was inhibited. TUNEL method showed the statistically significant difference of apoptotie index between the ad- p27mt group and the control group （48.5 ± 3.6, 4.2 ± 0.8, P 〈 0.01）. Conclusion： The above - mentioned human p27mt gene may cause malignant melanoma cell cycle to be arrested in G1 phase and subse- quently lead to apnptosis.