| 朱传东,王礼学,王国相,魏娟,张启佳,徐瀚峰,童金龙,郑勤.乏氧诱导的A549细胞外泌体特征及其对放射抗拒性的影响[J].中华放射医学与防护杂志,2018,38(8):567-573 |
| 乏氧诱导的A549细胞外泌体特征及其对放射抗拒性的影响 |
| The characteristics of hypoxia-induced exosomes secreted from A549 cells and its effect on radioresistance |
| 投稿时间：2018-01-28 |
| DOI：10.3760/cma.j.issn.0254-5098.2018.08.002 |
| 中文关键词: 外泌体 细胞外囊泡 乏氧 微环境 放射抗拒性 |
| 英文关键词:Exosomes Extracellular vesicles Hypoxia Microenvironment Radiation resistance |
| 基金项目:江苏省自然科学基金青年基金（BK20170134） |
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| 中文摘要: |
| 目的 收集乏氧和常氧条件下人肺腺癌A549细胞产生的外泌体，观察常氧下A549细胞与外泌体共培养后对X射线敏感性及侵袭性的变化。方法 分别在乏氧（1% O2）和常氧条件下（21% O2）培养A549细胞，超高速离心法收集在细胞分泌的常氧外泌体（N-EXO）和乏氧外泌体（H-EXO）。NanoSight检测外泌体数量，扫描电镜观察外泌体的外观和大小。蛋白印迹检测外泌体蛋白CD63。CCK8检测细胞对X射线的耐受性。荧光显微镜观察A549细胞对PKH67标记的外泌体的摄取。细胞划痕实验和Transwell实验观察加入不同的外泌体后细胞侵袭和侵袭性变化，ELISA方法检测细胞上清液中基质金属蛋白酶2（MMP2）和基质金属蛋白酶9（MMP9）的表达变化。细胞克隆形成实验观察二种外泌体对细胞放射抵抗性的改变。结果 每ml细胞培养液中H-EXO的数量增多。H-EXO和N-EXO均呈现出典型的环饼状，H-EXO直径变小，粒径分布在30~200 nm，小于N-EXO的粒径（50~220 nm）。H-EXO和N-EXO的CD63表达无明显差别。乏氧条件培养的A549细胞接受2 Gy X射线后，细胞的增殖水平在4和6 d时远高于常氧条件下的细胞。PKH67绿色荧光标记的外泌体分布在细胞内部。12、24、48 h后，H-EXO组细胞划痕宽度远小于N-EXO组（t=2.96、6.76、3.35，P<0.05）。H-EXO组细胞穿膜细数量大于N-EXO组和对照组（t=4.84、7.88，P<0.01）。H-EXO组上清液中MMP2（t=4.70、3.21，P<0.05）和MMP9（t=5.61、3.76，P<0.05）表达水平较对照组和N-EXO组均明显增高。对照组、N-EXO和H-EXO的D0值分别为2.614、2.552、4.850。H-EXO较N-EXO可以明显增强A549细胞的放射抵抗性。结论 乏氧条件下，A549细胞释放的外泌体数量增多、粒径变小，乏氧外泌体可以促进常氧细胞的侵袭性，并且能够增强细胞对X射线的耐受性。 |
| 英文摘要: |
| Objective To investigate the effect of exosomes secreted from human lung adenocarcinoma A549 cells under hypoxic or normoxic conditions on the radiosensitivity and invasiveness of normoxia cells.Methods A549 cells were cultured in hypoxic (1% O2) and normoxic (21% O2) conditions, respectively. The exosomes (N-EXO and H-EXO) secreted from normoxic or hypoxic A549 cells were collected by ultracentrifugation and its number was measured using a NanoSight detector. The appearance and size distribution of exosomes were observed by a scanning electron microscopy. The exosomal marker protein CD63 was measured by Western blot. The proliferation of cells exposed to X-rays under hypoxic or normoxic conditions were detected by CCK8 assay. The cell uptake situation of exosomes labeled with PKH67 was observed by a fluorescence microscopy. Cell migration and invasiveness were detected by a cell scratch test and transwell assay. The expression of matrix metalloproteinase 2 (MMP2) and MMP9 was detected by ELISA. Cellular radioresistance effect of exosomes was evaluated by a colony formation assay.Results The NanoSight measurement showed the number of exosomes in cell culture medium was increased after hypoxia treatment. The H-EXO and N-EXO showed typical ring cake shape. The size distribution of H-EXO was mainly between 30 nm and 200 nm, smaller than that of N-EXO (50-220 nm). Western blot assay showed that CD63 was expressed in both H-EXO and N-EXO. At 4 and 6 days after 2 Gy X-rays irradiation, cell proliferation rate of hypoxia A549 cells was significantly higher than that of normoxia cells. The green fluorescent marker of exosomes, PKH67, was distributed inside of the cell. Cell scratch test showed that the width of H-EXO group was much smaller than that of N-EXO group at 12, 24 and 48 hours after exosomes treatment (t=2.96, 6.76, 3.35, P<0.05). Transwell assay showed that the number of transmembrane cells in the H-EXO group was more than that in the N-EXO group and the control group (t=4.84, 7.88,P<0.01). The expression levels of MMP2 (t=4.70, 3.21, P<0.05) and MMP9 (t=5.61, 3.76, P<0.05) in the supernatant of H-EXO group were significantly higher than those in the control and N-EXO groups. Cell survival assay showed that the D0 values of control, N-EXO and H-EXO group were 2.614, 2.552 and 4.50 respectively, indicating that H-EXO could enhance radioresistance of A549 cells significantly.Conclusions This study finds that the number of exosomes released from A549 cells was increased under hypoxic condition but its size becomes smaller than that under normoxia. Hypoxic exosomes can promote the migration of normoxia cells and enhance cell radioresistance as well. |
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