H1299旁效应细胞对X-射线辐射的适应性与TGF-β1通路相关

The adaptive response to X-ray irradiation in H1299 bystander cells is associated with TGF-β1 pathway

本研究探索辐射诱导人非小细胞肺癌H1299旁效应细胞的适应性反应及TGF-β1通路在其中的作用。采用培养液转移法得到辐射诱导的旁效应细胞,用克隆形成实验检测旁效应细胞受照射后的适应性反应,用Western Blotting检测旁效应细胞中SOD2的表达变化。结果发现:用条件培养液培养H1299旁效应细胞,并不影响细胞的克隆存活率;但是细胞经1 h和18 h未照射条件培养液培养后再接受2Gy的X-射线照射,其细胞存活率较培养于新鲜培养液的细胞受照后的存活率分别增加了12%和38%,经1 h和18 h照射条件培养液培养后的细胞再接受2Gy的X-射线照射,其细胞存活力在此基础上又分别增加了10%;当在照前用TGF-βR1抑制剂SB431542处理信号细胞后,旁效应细胞的适应性反应不再发生;而将SB431542直接加入条件培养液中,并未影响1 h条件培养液诱导的适应性,但是用含有SB431542的18 h未照射和照射条件培养液培养过的旁效应细胞经2 Gy照射后,其克隆存活率较未加SB431542组分别降低了28%和18%,18 h未照射和照射条件培养液组间差异却增大至24%;旁效应细胞经照射条件培养液培养3 h或5 h后其SOD2表达下降。以上结果表明经条件培养液培养后旁效应细胞对X-射线照射产生了适应性反应,照射条件培养液与未照射条件培养液相比,进一步增加了这种适应性,并且TGF-β1信号通路对该适应性有重要调控作用,而SOD2可能未参与这个过程。

The aim is to investigate the adaptive response in radiation-induced bystander H1299 non-small cell lung cancer cells and the potential roles TGF-13 1 pathway plays in the response. Medium transfer method was used to obtain radiation-induced bystander cells; clonogenic assay was used to observe the adaptive response in bystander cells after irradiation; Western Blotting was used to detect the alteration of SOD2 expression in bystander cells. The data show that the conditioned medium collected from both unirradiated and irradiated cells did not cause any change in the clonogenic cell survival of bystander cells. However, when bystander cells were irradiated with 2 Gy of X-rays after being cultured in 1 h or 18 h non-radiation conditioned medium, its clonogenic cell survival was 12% or 38% greater than that cultured in fresh medium. The clonogenic cell survival of cells irradiated with 2 Gy of X-rays after culture in 1 h or 18 h radiation conditioned medium was 10% greater than that cultured in relative non-radiation conditioned medium. Furthermore, when the signaling cells were pretreated with a TGF- βR1 inhibitor, SB431542, the adaptive response in bystander cells no longer occurred. But adding SB431542 into the conditioned medium caused more complex consequence. On one hand, it did not affect the adaptive response in bystander cells cultured in the conditioned medium harvested 1 h post irradiation; on the other hand, while it decreased the adaptive response in bystander ceils cultured in the non-radiation or radiation conditioned medium harvested 18 h post irradiation by 28% or 18% compared with that without SB431542, it increased the difference of the levels of adaptive response in bystander ceils between cultured in radiation conditioned and in non-radiation conditioned medium from I0% to 24%. Moreover, the expression of SOD2 of bystander cells decreased after culture in radiation-conditioned medium for 3 h and 5 h compared with that cultured in non-radiation-conditioned medium. The results suggested that the conditioned medium from unirradiated cells induced an adaptive response to X-irradiation in bystander cells, and that from irradiated cells caused a greater adaptive response. TGF-β1 pathway played an important mediating role in the occurrence of the response, but SOD2 was not involved.