| 李辰,田梅,苟巧,齐雪松,苏旭.缝隙连接蛋白43在受照人血管内皮细胞中的改变及其对受照细胞刚性的作用[J].中华放射医学与防护杂志,2021,41(6):418-425 |
| 缝隙连接蛋白43在受照人血管内皮细胞中的改变及其对受照细胞刚性的作用 |
| Changes of connexin 43 in irradiated human vascular endothelial cells and its influence on cell stiffness |
| 投稿时间：2020-12-30 |
| DOI：10.3760/cma.j.issn.0254-5098.2021.06.004 |
| 中文关键词: X射线 HUVEC细胞 Cx43 刚性 |
| 英文关键词:X-rays HUVEC cell Cx43 Stiffness |
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| 中文摘要: |
| 目的 研究X射线对人脐静脉内皮细胞（HUVEC）中缝隙连接蛋白43（Cx43）的表达、分布和细胞刚性的影响，初步探讨Cx43对受照细胞刚性的调控作用。方法 采用Western blot方法检测10 Gy X射线照射后不同时间（0、6、12、24和48 h）和不同剂量X射线（0、2.5、5、10和20 Gy）照射后12 h HUVEC细胞中Cx43表达水平的改变，以及不同剂量（0、5和10 Gy） X射线照射后不同时间（3、6、24和48 h） Cx43 3个磷酸化位点（Ser279/282、Ser368和Tyr265）磷酸化水平的变化。采用细胞免疫荧光方法检测Cx43蛋白在受照HUVEC细胞中的分布变化。采用原子力显微镜检测受照细胞在探针压入不同深度（50、100和200 nm）时杨氏模量（细胞刚性）的变化，以及Cx43过表达对受照细胞杨氏模量（细胞刚性）的影响。结果 10 Gy X射线照射后6、12、24、48 h，HUVEC细胞中Cx43表达量降低（t=3.262、3.708、3.686、6.825，P<0.05），且在2.5、5、10和20 Gy照射后24 h Cx43表达水平的降低与受照剂量呈现剂量依赖性（t=3.034、10.720、13.130、13.650，P<0.05）。5、10和20 Gy X射线照射后24 h，HUVEC细胞中Cx43分布由细胞间隙转移入核及核周，照射后24和48 h，Cx43的Ser368位点磷酸化水平升高并且随剂量增加而增加。10 Gy X射线照射HUVEC细胞后24 h，照射组与对照组相比，在探针压入100和200 nm时，杨氏模量均明显降低（t=3.362、5.122，P<0.05）；过表达Cx43的受照组与空载体受照组相比，在探针压入100和200 nm时，杨氏模量增高（t=2.674、4.398，P<0.05）。结论 X射线照射可导致HUVEC细胞内Cx43 Ser368位点磷酸化，促进Cx43降解和分布变化，降低细胞刚性。提高Cx43的表达水平，有助于受照细胞刚性的恢复，提示Cx43可能是调控X射线致血管内皮细胞损伤的作用靶点。 |
| 英文摘要: |
| Objective To investigate the changes of connexin 43 (Cx43) in human umbilical vein endothelial cells (HUVEC) after X-ray irradiation and its influence on the stiffness of irradiated cells. Methods Western blot was used to detect the expression of Cx43 in HUVEC cells at different time points (0,6,12,24 and 48 h) after different doses of X-ray irradiation (0, 2.5, 5, 10 and 20 Gy), and the phosphorylation levels of three phosphorylation sites (Ser279/282, Ser368 and Tyr265) of Cx43 at different time points (3, 6, 24 and 48 h) after 0, 5 and 10 Gy irradiation. The distribution of Cx43 protein in the irradiated HUVEC cells was detected by immunofluorescence. The stiffness changes of cells were detected by atomic force microscopy (AFM) at the depths of 50, 100 and 200 nm. Results The expression of Cx43 in HUVEC cells was reduced at 6, 12, 24 and 48 h after 10 Gy X-ray irradiation(t=3.262,3.708,3.686,6.825, P<0.05) and this decrease had a dose dependent manner at 24 h after 2.5, 5, 10 and 20 Gy irradiation (t=3.034, 10.720, 13.130, 13.650, P<0.05). At 24 h after 5, 10 and 20 Gy X-ray irradiation, the distribution of Cx43 in HUVEC cells was transported from intercellular gap junctions to nucleus and perinuclear region. At 24-48 h after irradiation, the phosphorylation level of Ser368 at Cx43 increased and in a dose dependent manner. At 24 h after irradiation, the stiffness of the irradiated cells decreased significantly under the conditions of 100 and 200 nm (t=3.362, 5.122, P<0.05), and recovered with overexpression of Cx43 (t=2.674,4.398,P<0.05). Conclusions X-ray irradiation leads to the phosphorylation of Ser368 at Cx43, which promotes the degradation and nucleus/perinuclear translocation of Cx43 and reduces the stiffness of HUVEC. Increasing the expression level of Cx43 is helpful to the stiffness recovery of irradiated vascular endothelial cells, suggesting that Cx43 may be a potential target for regulating radiation injury of vascular endothelial cells. |
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