X线照射人肺腺癌细胞后侵袭和转移能力变化及机制探讨

Changes in abilities of invasion and metastasis in surviving human lung adenocarcinoma （ A549 ） cell safter X-ray irradiation and relatedmechanism

目的探讨X线照射后存活的人肺腺癌（A549）细胞侵袭和转移能力变化及机制。方法用克隆形成实验检测X线照射前后放射敏感性变化，用MTT黏附实验、Transwell侵袭和迁移实验检测侵袭、转移能力。用SYBRGreen荧光定量RT-PCR及蛋白印记法检测E-Cadherin、Vimentin、TGF-β1、MMP-2、MMP-9的mRNA及蛋白表达变化来探讨机制。结果克隆形成实验结果显示X线照射后存活细胞对放射更加耐受（Dn值比为0．94），其黏附、侵袭、迁移能力较照射前增加1．46、1．40、1．45倍。照射后细胞间隙增大，有多而长的伪足形成。照射后Vimentin、TGF-β1、MMP-2、MMP-9的mRNA表达均上调（为对照组的1．37、2．37、1．80、1．50倍），其蛋白表达也相应上调（为对照组的1．60、1．80、1．10、1．20倍），而E-Cadherin的mRNA及蛋白表达则下调（为对照组的59．4％、74．6％）。结论X线照射后A549细胞侵袭和转移能力较照射前明显增强，其机制可能与放射诱导TGF-β1表达进而促进上皮细胞问质化改变有关，还可能与放射诱导MMP-2、MMP-9表达增强有关。

Objective To investigate the changes in the abilities of invasion and metastasis in surviving human lung adenocarcinoma （A549 ） cells after X-ray irradiation and the related mechanism. Methods The change in radiosensitivity after X-ray irradiation was determined by colony formation assay. The abilities of invasion and metastasis were evaluated by MTY adhesion assay and Transwell invasion and migration assay in vitro. The mRNA and protein expression of E-cadhcrin, vimentin, tumor growth factor （TGF）-β1, matrix metalloproteinase （MMP）-2, and MMP-9 was measured by real-time RT-PCR using SYBR Green fluorescence and Western blot. Results The colony formation assay showed that the surviving A549 cells after X-ray irradiation were more resistant to irradiation （ratio of Do values = 0. 94 ）. Their abilities of adhesion, invasion, and migration were significantly increased by 1.46 times, 1.40 times, and 1.45 times, respectively. In addition, the surviving cells showed enlarged intercellular spaces and had many long pseudopodi. Compared with those in the control group, the mRNA expression levels of vimentin, TGF- β1, MMP-2, and MMP-9 of surviving cells were increased by 1.37 times, 2. 37 times, 1.80 times, and I. 50 times, respectively, the protein expression levels of the above substances were increased by 1.60 times, 1.80 times, 1.10 times, and 1.20 times, respectively, and the mRNA and protein expression levels of E-cadherin were decreased by 59.4% and 74. 6%. Conclusions The surviving A549 cells after X-ray irradiation have significantly increased abilities of invasion and metastasis. This may be due to radiation- induced TGF-β1 expression increase that in turn promotes epithelial-mesenchymal transition and also clue to radiation-induced MMP-2 and MMP-9 expression increase.