腺病毒介导的PTEN对A549肺癌细胞体内外生长的影响

Influence of adenovirus mediated PTEN gene on growth of A549 cells in vitro and in vivo

目的:研究携带第10号染色体缺失的磷酸脂酶和张力蛋白同源基因(phosphatase and tensin homologue-deleted chromosome ten gene,PTEN)的腺病毒表达载体对A549肺癌细胞体内外生长的抑制作用。方法:从人外周血淋巴细胞中通过RT-PCR扩增出PTEN基因片段,将其克隆到pAdTrack-CMV转移载体上,构建了PTEN重组腺病毒载体。体外用MTT法检测Ad-PTEN对A549肺癌细胞生长的抑制作用,以流式细胞术检测肿瘤细胞的周期和凋亡率。建立荷瘤裸鼠模型,体内检测Ad-PTEN对A549肺癌细胞移植瘤生长的影响,以免疫组化法检测移植瘤中微血管密度。结果:克隆的PTEN基因测序结果与基因Bank数据库完全相符。Ad-PTEN体外感染A549肺癌细胞48h后其凋亡率为10.5%,明显高于对照细胞;4d后细胞生长与对照组细胞相比抑制了57%。肺癌细胞移植瘤治疗结束时,Ad-PTEN组瘤重为(0.58±0·29)g,对照组瘤重为(1.42±0.24)g,其生长抑制达59%(P<0.05);同时移植瘤中微血管密度降低约49%(P<0.05)。结论:成功构建的Ad-PTEN腺病毒载体能在体内外抑制A549肺癌细胞及其移植瘤的生长。

Objective： To study the inhibitory effect of adenovirus mediated （phosphatase and tensin homologuedeleted chromosome ten gene） PTEN gene on the growth of A549 cells in vitro and in vivo. Methods： The PTEN gene was amplified by RT-PCR from human peripheral blood and was inserted into pAdTrack-CMV to construct pAdTrack-CMV-PTEN. The growth inhibition effect of pAdTrack-CMV-PTEN on A549 cells was detected by MTT; flow cytometry was used to detect the cell cycle and apoptosis of the tumor cells. The tumor inhibitory effect of pAdTrack-CMV-PTEN on A549 cells was studied with tumor-bearing mice model. The microvessel density （MVD） of the implanted tumor tissue was determined immunohisto- chemically. Results： The sequencing result of cloned PTEN accorded well to that in GeneBank. The apoptosis rate of A549 cells was 10.5% 48 h after infection with pAdTrack-CMV-PTEN, significantly higher than that in the control group （0）. Four days after infection, the growth of the A549 was inhibited by 57%. At the end of tumor inhibition experiment, the average tumor weight was （0.58 ± 0.29） g in pAdTrack-CMV-PTEN treated group and （1.42 ± 0.24） g in the control group, with a inhibitory rate of 59% （ P 〈 0.05 ） ; pAdTrack-CMV-PTEN also decreased MVD by 49% （ P 〈 0. 05 ）. Conclusion ： We have sucessfully constructed pAdTrack-CMV-PTEN, which can inhibit the growth of A549 cells in vitro and in vivo.