| 孙嘉锴,张家玮,李 鲲,郑 峤,宋基伟,梁卓文,胡学昱,王 哲.810nm弱激光作用下巨噬细胞对星形胶质细胞的影响[J].中国脊柱脊髓杂志,2019,(2):156-163. |
| 810nm弱激光作用下巨噬细胞对星形胶质细胞的影响 |
| 中文关键词: 脊髓损伤 弱激光治疗 M1型巨噬细胞 星形胶质细胞 硫酸软骨素蛋白多糖 |
| 中文摘要: |
| 【摘要】目的：探讨810nm弱激光作用下巨噬细胞对星形胶质细胞活化及硫酸软骨素蛋白多糖（Chondroitin sulfate proteoglycans，CSPG）分泌的影响。方法：构建脊髓损伤（spinal cord injury，SCI）动物模型，设立脊髓损伤（SCI）组和脊髓损伤后弱激光治疗（low level laser therapy，LLLT）组，免疫荧光检测脊髓损伤区星形胶质细胞标志物胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）和CSPG的表达情况；建立体外巨噬细胞-星形胶质细胞共培养模型，分为Ctrl组（空白对照），M1组（采用M1型巨噬细胞条件培养基培养星形胶质细胞）；M1＋LLLT组（采用M1条件培养基培养星形胶质细胞，同时进行弱激光照射），采用Western Blot检测M1型巨噬细胞标志物诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）和星形胶质细胞活化经典通路信号传导及转录激活因子3（signal transducers and activators of transcription，STAT3）的表达，细胞计数试剂盒（cell counting kit-8，CCK-8）法检测星形胶质细胞的活性，实时定量PCR（real-time quantitative PCR，RT-qPCR）和酶联免疫吸附实验（enzyme-linked immunosorbent assay，ELISA）检测CSPG的核心蛋白聚集蛋白聚糖（aggrecan）和多功能蛋白聚糖（versican）的表达情况。结果：体内实验发现，LLLT组小鼠脊髓损伤区GFAP和CSPG表达较SCI组均减少（P<0.05）。体外实验结果显示，LLLT后巨噬细胞iNOS的表达显著降低（P<0.05）。与Ctrl组相比，M1组星形胶质细胞的活性明显升高（P<0.05），磷酸化信号转导与转录激活因子3（phosphorylated-signal transduction and activators of transcription 3，pSTAT3）的表达显著增加（P<0.05）。而相较于M1组，M1+LLLT组星形胶质细胞活性显著下降（P<0.05），pSTAT3/STAT3的表达明显抑制（P<0.05）。RT-qPCR及ELISA结果显示，M1组星形胶质细胞aggrecan、versican的mRNA和蛋白的表达比Ctrl组均显著增强（P<0.05）；M1+LLLT组相较于M1组，aggrecan的mRNA和蛋白的表达受到抑制（P<0.05），versican的蛋白表达虽有下降，但无统计学差异。结论：LLLT可抑制小鼠脊髓损伤区星形胶质细胞的活化，减少CSPG的表达，且这种作用可通过弱激光作用下的巨噬细胞实现。 |
Effect of macrophages on astrocytes under 810nm low level laser therapy |
| 英文关键词:Spinal cord injury Low level laser therapy M1 macrophage Astrocyte Chondroitin sulfate proteoglycans |
| 英文摘要: |
| 【Abstract】 Objectives: To investigate the effects of macrophages controlled by 810nm low level laser on the activation of astrocytes and the secretion of chondroitin sulfate proteoglycans(CSPG). Methods: An animal model of spinal cord injury was established, including spinal cord injury group(SCI) and low level laser therapy group(LLLT). Immunofluorescence was used to detect the expression of glial fibrillary acidic protein (GFAP) and CSPG in spinal cord injury area. The macrophage-astrocyte model was established in vitro, which was divided into Ctrl group(blank control), M1 group(astrocytes were cultured in M1 conditioned medium) and M1+LLLT group(astrocytes were cultured in M1 conditioned medium with low level laser), Western Blot was used to detect the expression of M1 marker inducible nitric oxide synthase(iNOS) and astrocyte activation pathway signaling and activators of transcription(STAT3). The activity of astrocytes was detected by CCK-8. RT-qPCR and ELISA were used to assay the expression of the core protein of CSPG(aggrecan and versican). Results: In vivo experiments showed that the expression of GFAP and CSPG in the spinal cord injury area in LLLT group was lower than that in SCI group(P<0.05). The results of in vitro experiments showed that the expression of iNOS in macrophages significantly decreased(P<0.05) after LLLT. The activity of astrocytes in M1 group was significantly increased, compared with the Ctrl group(P<0.05), and the expression of pSTAT3/STAT3 significantly increased(P<0.05). Compared with M1 group, the activity of astrocytes in M1+LLLT group significantly decreased(P<0.05), and the expression of pSTAT3 was significantly inhibited(P<0.05). RT-qPCR and ELISA showed the expression of aggrecan and versican mRNA and protein in M1 group was significantly higher than that in Ctrl group(P<0.05). Compared with M1 group, the experession of aggrecan mRNA and protein in M1+LLLT group was inhibited(P<0.05). However, while the protein expression of versican decreased, but there was no statistical difference. Conclusions: Low level laser therapy can inhibit the activation of astrocytes and reduce the expression of CSPG in the spinal cord injury region of SCI mice, which can be achieved by macrophages under low level laser. |
| 投稿时间：2018-10-23 修订日期：2018-11-27 |
| DOI： |
| 基金项目:国家自然科学基金（81572151），陕西省社会发展攻关计划项目（2016SF-143） |
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